Glycotargeting therapeutics

ABSTRACT

Glycotargeting therapeutics are useful in the treatment of transplant rejection, autoimmune disease, food allergy, and immune response against a therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of co-pending U.S. application Ser.No. 16/723,774, filed Dec. 20, 2019, which is a continuation of U.S.application Ser. No. 14/627,297, filed Feb. 20, 2015, now U.S. Pat. No.10,821,157, which claims the benefit of priority to U.S. Ser. No.61/942,942 filed Feb. 21, 2014, the disclosures of each of which arehereby incorporated by reference in their entireties.

FIELD OF THE DISCLOSURE

The field of the disclosure relates to pharmaceutically acceptablecompositions that are useful in the treatment of transplant rejection,autoimmune disease, food allergy, and immune response against atherapeutic agent.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the material contained in theSequence Listing XML file being submitted concurrently herewith: Filename: ANOK002C2_ST26.xml; created Sep. 7, 2022, 56747 bytes in size.

BACKGROUND OF THE DISCLOSURE

Applications US 2012/0039989, US 2012/0178139 and WO 2013/121296describe the targeting of antigens to erythrocytes to take advantage ofthe erythrocytes' role in antigen presentation for tolerization.Notwithstanding the positive results generated to date with thisapproach, the possibility of alternative approaches has remained ofinterest.

SUMMARY OF THE DISCLOSURE

An aspect of the disclosure provides a composition comprising a compoundof Formula 1:

X

Y—Z]_(m)   Formula 1

where:

-   -   m is an integer from about 1 to 100;    -   X comprises an antigen against which a patient develops an        unwanted immune response, or a tolerogenic portion thereof; or    -   X comprises an antibody, antibody fragment or ligand that        specifically binds a circulating protein or peptide or antibody,        which circulating protein or peptide or antibody is causatively        involved in transplant rejection, immune response against a        therapeutic agent, autoimmune disease, hypersensitivity and/or        allergy;    -   Y comprises a linker moiety; and    -   Z comprises a liver-targeting moiety.

Z can also comprise galactose, galactosamine or N-acetylgalactosamine,for example, conjugated at its C1, C2 or C6 to Y.

Y can be Y is selected from N-hydroxysuccinamidyl linkers, malaemidelinkers, vinylsulfone linkers, pyridyl di-thiol-poly(ethylene glycol)linkers, pyridyl di-thiol linkers, n-nitrophenyl carbonate linkers,NHS-ester linkers, and nitrophenoxy poly(ethylene glycol)ester linkers.

Y can also comprise: an antibody, antibody fragment, peptide or otherligand that specifically binds X; a disulfanyl ethyl ester; a structurerepresented by one of Formulae Ya to Yp:

or has a portion represented by Formula Y′-CMP:

where: the left bracket “(“indicates the bond between X and Y; the rightor bottom bracket and”)” indicates the bond between Y and Z; n is aninteger from about 1 to 100; p is an integer from about 2 to 150; q isan integer from about 1 to 44; R⁸ is —CH₂— or —CH₂—CH₂—C(CH₃)(CN)—; R⁹is a direct bond or —CH₂—CH₂—NH—C(O)—; and Y′ represents the remainingportion of Y.

In another aspect of the above, n is about 40 to 80, p is about 10 to100, q is about 3 to 20, R⁸ is —CH₂—CH₂—C(CH₃)(CN)—; and when R⁹ is—CH₂—CH₂—NH—C(O)—, Z is galactose or N-acetylgalactosamine conjugated atits C1.

In still another aspect of the above, Y comprises Formula Ya, FormulaYb, Formula Yh, Formula Yi, Formula Yk, Formula Ym or Formula Yn,particularly Formula Ya, Formula Yb, Formula Ym or Formula Yn.

X can further comprise: a foreign transplant antigen against whichtransplant recipients develop an unwanted immune response; a foreignfood, animal, plant or environmental antigen against which patientsdevelop an unwanted immune response; a foreign therapeutic agent againstwhich patients develop an unwanted immune response; or a syntheticself-antigen against the endogenous version of which patients develop anunwanted immune response, or a tolerogenic portion thereof.

The disclosure also pertains to a method of treatment for an unwantedimmune response against an antigen by administering to a mammal in needof such treatment an effective amount of a composition comprising acompound of Formula 1 as discussed above. In such method the compositioncan be administered for clearance of a circulating protein or peptide orantibody that specifically binds to antigen moiety X, which circulatingprotein or peptide or antibody is causatively involved in transplantrejection, immune response against a therapeutic agent, autoimmunedisease, hypersensitivity and/or allergy. The composition can beadministered in an amount effective to reduce a concentration of theantibodies that are causatively involved in transplant rejection, immuneresponse against a therapeutic agent, autoimmune disease,hypersensitivity and/or allergy in blood of the patient by at least 50%w/w, as measured at a time between about 12 to about 48 hours after theadministration. The composition can administered for tolerization of apatient with respect to antigen moiety X.

Yet another aspect of the disclosure provides a composition comprising acompound of Formula 2:

where: m′ is zero or an integer from about 1 to 10, m″ is zero or aninteger from about 1 to 10, and the sum of m′+m″ is an integer fromabout 1 to 10 but is at least 1; each X is a foreign antigen orself-antigen against which a patient develops an unwanted immuneresponse, or a tolerogenic portion thereof; each Y is a linker moiety ora direct bond, or an antibody, antibody fragment, peptide or otherligand that specifically binds X; and Z is a liver-targeting moiety,provided that X is not interferon, Ribavirin, Nexavar/Sorafenib,Erbitus/Cetuximab, Avastatin/bevacizumab or Herceptin/trastuzumab whenm′+m″ equals 1 and Z is DOM 26h-196-61

In the above aspect involving Formula 2, Z can comprise anASGPR-targeted antibody, an ASGPR-targeted antibody fragment, anASGPR-targeted peptide, an ASGPR-targeted scFv, or another ASGPR ligand.Y can be a linker having an immunoproteosome cleavage site. X cancomprise a group of related antigens against which a patient develops anunwanted immune response or a group of tolerogenic fragments thereof.For example, X can be selected from the groups comprising:

-   -   two or more of insulin, proinsulin, preproinsulin, glutamic acid        decarboxylase-65 GAD-67, glucose-6 phosphatase 2,        insulinoma-associated protein 2, insulinoma-associated protein        2β, ICA69, ICA12, carboxypeptidase H, Imogen 38, GLIMA 38,        chromogranin-A, HSP-60, caboxypeptidase E, peripherin, glucose        transporter 2, hepatocarcinoma-intestine-pancreas/pancreatic        associated protein, S100β, glial fibrillary acidic protein,        regenerating gene II, pancreatic duodenal homeobox 1, dystrophia        myotonica kinase, islet-specific glucose-6-phosphatase catalytic        subunit-related protein, and SST G-protein coupled receptors        1-5;    -   two or more of myelin basic protein, myelin oligodendrocyte        glycoprotein, myelin proteolipid protein, SEQ ID NO:11, SEQ ID        NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,        and SEQ ID NO:17; and    -   two or more of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ        ID NO:27.

The disclosure also pertains to a method of treatment for an unwantedimmune response against an antigen by administering to a mammal in needof such treatment an effective amount of a composition comprising acompound of Formula 2 as discussed above. In such method the compositioncan be administered for clearance of a circulating protein or peptide orantibody that specifically binds to antigen moiety X, which circulatingprotein or peptide or antibody is causatively involved in transplantrejection, immune response against a therapeutic agent, autoimmunedisease, hypersensitivity and/or allergy. The composition can beadministered in an amount effective to reduce a concentration of theantibodies that are causatively involved in transplant rejection, immuneresponse against a therapeutic agent, autoimmune disease,hypersensitivity and/or allergy in blood of the patient by at least 50%w/w, as measured at a time between about 12 to about 48 hours after theadministration. The composition can administered for tolerization of apatient with respect to antigen moiety X.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing galactose conjugation (F1aA-PE-m₄-n₈₀(Gal-PE)) preferentially targets OVA to sinusoidal endothelia cells ofthe liver (LSECs).

FIG. 1B is a graph showing galactose conjugation (F1aA-PE-m₄-n₈₀(Gal-PE)) preferentially targets OVA to Kupffer cells of the liver (KC).

FIG. 1C is a graph showing galactose conjugation (F1aA-PE-m₄-n₈₀(Gal-PE)) preferentially targets OVA to hepatocytes of the liver.

FIG. 1D is a graph showing galactose conjugation (F1aA-PE-m₄-n₈₀(Gal-PE)) preferentially targets OVA to other antigen-presenting cellsof the liver (APCs).

FIG. 2 is a graph showing proliferation of OTI CD8+ T cells in micetreated with F1aA-OVA-m₄-n₈₀ (Gal-OVA), OVA or saline (i.e. naïve).

FIG. 3A is a graph showing the percentage of OT-1 CD8⁺ T cellspresenting surface markers PD-1+ in generations of proliferating T cellstreated with saline, OVA or F1aA-OVA-m₄-n₈₀ (GAL-OVA).

FIG. 3B is a graph showing the percentage of OT-1 CD8⁺ T cellspresenting surface markers Annexin-V+ in generations of proliferating Tcells treated with saline, OVA or F1aA-OVA-m₄-n₈₀ (GAL-OVA).

FIG. 4 is a graph showing galactose conjugation (F1aA-OVA-m₄-n₈₀(Gal-OVA)) decreases the immunogenicity of OVA as determined byOVA-specific antibody titers (shown in Ab titers log⁻¹).

FIG. 5 shows that F1aA-OVA-m₄-n₈₀ (Gal-OVA) is able to depleteOVA-specific antibodies from the serum.

FIG. 6 shows that F1aA-OVA-m₄-n₈₀ (mGal-OVA), F1b-OVA-m₁-n₄₄-p₃₄(pGal-OVA), and N′-DOM-Gly3Ser-OVA-Gly3Ser-6×His-C′ (Dom-OVA) are ableto mitigate the OVA-specific immune response in draining lymphnodesafter intradermal challenge with OVA and the andjuvant LPS.

FIG. 7A shows the characterization of F1aA-OVA-m₄-n₈₀ andF1b-OVA-m₁-n₄₄-p₃₄ by Size-exclusion HPCL traces of F1aA-OVA-m₄-n₈₀(magenta), F1b-OVA-m₁-n₄₄-p₃₄ (blue) and unconjugated OVA (black). Shiftto the left represents an increase in molecular weight.

FIG. 7B shows the characterization of F1aA-OVA-m₄-n₈₀ andF1b-OVA-m₁-n₄₄-p₃₄ by Polyacrylamide gel demonstrating increasedmolecular weight after OVA conjugation: (1.) Unconjugated OVA, (2.)F1aA-OVA-m₄-n₈₀ and (3.) F1b-OVA-m₁-n₄₄-p₃₄.

FIG. 8 is a graph showing the normalized quantity ofN-OVA-Gly₃Ser-6×His-C(OVA), N-DOM-Gly₃Ser-OVA-Gly3Ser-6×His-C (DOM-OVA),or N-OVA-Gly₃Ser-DOM-Gly₃Ser-6×His-C(OVA-DOM) in the circulation of miceafter injection as an i.v. bolus.

FIG. 9A is a series of graphs showing the titer of anti-OVA IgGantibodies in the circulation of individual mice after treatment withsaline, DOM and OVA, DOM-OVA, or OVA-DOM. Production of anti-OVA IgG wasinduced by i.v. injections of OVA alone. Treatment times are indicatedby vertical dashed lines. Titer is calculated as log₁₀ of the maximalfold dilution of plasma with detectable anti-OVA IgG.

FIG. 9B is a series of graphs showing the titer of anti-OVA IgGantibodies in the circulation of individual mice after treatment withsaline, DOM and OVA, DOM-OVA, or OVA-DOM. Production of anti-OVA IgG wasinduced by i.v. injections of OVA and CpG-B. Treatment times areindicated by vertical dashed lines. Titer is calculated as logic of themaximal fold dilution of plasma with detectable anti-OVA IgG.

DETAILED DESCRIPTION

The two known asialoglycoprotein receptors (“ASGPRs”) are expressed onhepatocytes and liver sinusoidal endothelial cells (or “LSECs”). Othergalactose/galactosamine/N-acetylgalactosamine receptors can be found invarious forms on multiple cell types [e.g., dendritic cells,hepatocytes, LSECs, and Kupffer cells]. Dendritic cells are considered“professional antigen presenting cells,” because their primary functionis to present antigens to the immune system for generating immuneresponses. Some cells within the liver are known to be able to presentantigens, but the liver is more known to be involved in tolerogenesis.The liver is understood to be a tolerogenic organ. For example, lowerincidences of rejection are reported in cases of multiple organtransplants when the liver is one of the organs transplanted. LSECs aremuch newer to the literature; consequently their role in tolerogenesisand/or moderation of inflammatory immune responses is not yet widelyacknowledged or well understood. However, it is becoming clear that theyalso can play a significant role in the induction of antigen-specifictolerance.

One of the distinctive features of the erythrocyte surface is itsglycosylation, i.e., the presence of significant numbers of glycosylatedproteins. Indeed, the glycophorins (e.g., glycophorin A) have beenemployed as targets for erythrocyte binding. Glycophorins are proteinswith many covalently attached sugar chains, the end terminus of which issialic acid. As an erythrocyte ages and becomes ripe for clearance, theterminal sialic acid of its glycophorins tends to be lost, leavingN-acetylgalactosamine at the free end. N-acetylgalactosamine is a ligandselectively received by the ASGPR associated with hepatic cells, leadingto binding of N-acetylgalactosamine-containing substances by hepaticcells and their subsequent uptake and processing in the liver.

Heretofore, it has been understood by those skilled in the art thatglycosylation of a therapeutic agent in a manner that results in hepatictargeting should be avoided due to first-pass clearance by the liverresulting in poor circulation half-life of the therapeutic agent. By thesame token, some monoclonal antibodies need to be specificallyglycosylated at ASN297 for optimal binding to their Fc receptors. It hasnow surprisingly been found that galactosylation can be used in a mannerthat induces tolerogenesis.

The present disclosure provides certain therapeutic compositions thatare targeted for delivery to (and for uptake by) the liver, particularlyhepatocytes, LSECs, Kupffer cells and/or stellate cells, moreparticularly hepatocytes and/or LSECs, and even more particularly tospecifically bind ASGPR. Liver-targeting facilitates two mechanisms oftreatment: tolerization and clearance. Tolerization takes advantage ofthe liver's role in clearing apoptotic cells and processing theirproteins to be recognized by the immune system as “self,” as well as theliver's role in sampling peripheral proteins for immune tolerance.Clearance takes advantage of the liver's role in blood purification byrapidly removing and breaking down toxins, polypeptides and the like.Targeting of these compositions to the liver is accomplished by agalactosylating moiety (e.g., galactose, galactosamine andN-acetylgalactosamine, particularly conjugated at C1, C2 or C6), byanother liver-targeting moiety (e.g., a monoclonal antibody, or afragment or a scFv thereof), or by de-sialylating a polypeptide forwhich such liver-targeting is desired. The galactosylating or otherliver-targeting moiety can be chemically conjugated or recombinantlyfused to an antigen, whereas desialylation exposes a galactose-likemoiety on an antigen polypeptide. The antigen can be endogenous (aself-antigen) or exogenous (a foreign antigen), including but notlimited to: a foreign transplant antigen against which transplantrecipients develop an unwanted immune response (e.g., transplantrejection), a foreign food, animal, plant or environmental antigen towhich patients develop an unwanted immune (e.g., allergic orhypersensitivity) response, a therapeutic agent to which patientsdevelop an unwanted immune response (e.g., hypersensitivity and/orreduced therapeutic activity), a self-antigen to which patients developan unwanted immune response (e.g., autoimmune disease), or a tolerogenicportion (e.g., a fragment or an epitope) thereof; these compositions areuseful for inducing tolerization to the antigen. Alternatively, thegalactosylating or other liver-targeting moiety can be conjugated to anantibody, antibody fragment or ligand that specifically binds acirculating protein or peptide or antibody, which circulating protein orpeptide or antibody is causatively involved in transplant rejection,immune response against a therapeutic agent, autoimmune disease, and/orallergy (as discussed above); these compositions are useful for clearingthe circulating protein, peptide or antibody. Accordingly, thecompositions of the present disclosure can be used for treating anunwanted immune response, e.g., transplant rejection, an immune responseagainst a therapeutic agent, an autoimmune disease, and/or an allergy.Also provided are pharmaceutical compositions containing atherapeutically effective amount of a composition of the disclosureadmixed with at least one pharmaceutically acceptable excipient. Inanother aspect, the disclosure provides methods for the treatment of anunwanted immune response, such as transplant rejection, response againsta therapeutic agent, autoimmune disease or allergy.

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

The singular forms “a,” “an,” and “the” include plural referents, unlessthe context clearly indicates otherwise.

The term “about” when used in connection with a numerical value is meantto encompass numerical values within a range typically having a lowerlimit that is, e.g., 5-10% smaller than the indicated numerical valueand having an upper limit that is, e.g., 5-10% larger than the indicatednumerical value.

An “antigen” is any substance that serves as a target for the receptorsof an adaptive immune response, such as the T cell receptor, B cellreceptor or an antibody. An antigen may originate from within the body(“self,” “auto” or “endogenous”). An antigen may originate from outsidethe body (“non-self,” “foreign” or “exogenous”), having entered, forexample, by inhalation, ingestion, injection, or transplantation.Foreign antigens include, but are not limited to, food antigens, animalantigens, plant antigens, environmental antigens, therapeutic agents, aswell as antigens present in an allograft transplant.

An “antigen-binding molecule” as used herein relates to molecules, inparticular to proteins such as immunoglobulin molecules, which containantibody variable regions providing a specific binding to an epitope.The antibody variable region can be present in, for example, a completeantibody, an antibody fragment, and a recombinant derivative of anantibody or antibody fragment. The term “antigen-binding fragment” of anantibody (or “binding portion”), as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind atarget sequence. Antigen-binding fragments containing antibody variableregions include (without limitation) “Fv”, “Fab”, and “F(ab′)₂” regions,“single domain antibodies (sdAb)”, “nanobodies”, “single chain Fv(scFv)” fragments, “tandem scFvs” (V_(H)A-V_(L)A-V_(H)B-V_(L)B),“diabodies”, “triabodies” or “tribodies”, “single-chain diabodies(scDb)”, and “bi-specific T-cell engagers (BiTEs)”.

A “chemical modification” refers to a change in the naturally-occurringchemical structure of one or more amino acids of a polypeptide. Suchmodifications can be made to a side chain or a terminus, e.g., changingthe amino-terminus or carboxyl terminus. In some embodiments, themodifications are useful for creating chemical groups that canconveniently be used to link the polypeptides to other materials, or toattach a therapeutic agent.

The term “comprising”, which is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps. The phrase“consisting of” excludes any element, step, or ingredient not specified.The phrase “consisting essentially of” limits the scope of describedsubject matter to the specified materials or steps and those that do notmaterially affect its basic and novel characteristics.

“Conservative changes” can generally be made to an amino acid sequencewithout altering activity. These changes are termed “conservativesubstitutions” or mutations; that is, an amino acid belonging to agrouping of amino acids having a particular size or characteristic canbe substituted for another amino acid. Substitutes for an amino acidsequence can be selected from other members of the class to which theamino acid belongs. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, methionine, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such substitutions are notexpected to substantially affect apparent molecular weight as determinedby polyacrylamide gel electrophoresis or isoelectric point. Conservativesubstitutions also include substituting optical isomers of the sequencesfor other optical isomers, specifically D amino acids for L amino acidsfor one or more residues of a sequence. Moreover, all of the amino acidsin a sequence can undergo a D to L isomer substitution. Exemplaryconservative substitutions include, but are not limited to, Lys for Argand vice versa to maintain a positive charge; Glu for Asp and vice versato maintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free —NH₂. Yet another type ofconservative substitution constutes the case where amino acids withdesired chemical reactivities are introduced to impart reactive sitesfor chemical conjugation reactions, if the need for chemicalderivativization arises. Such amino acids include but are not limited toCys (to insert a sulfhydryl group), Lys (to insert a primary amine), Aspand Glu (to insert a carboxylic acid group), or specialized noncanonicalamino acids containing ketone, azide, alkyne, alkene, and tetrazineside-chains. Conservative substitutions or additions of free —NH₂ or —SHbearing amino acids can be particularly advantageous for chemicalconjugation with the linkers and galactosylating moieties of Formula 1.Moreover, point mutations, deletions, and insertions of the polypeptidesequences or corresponding nucleic acid sequences can in some cases bemade without a loss of function of the polypeptide or nucleic acidfragment. Substitutions can include, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues. A variant usable inthe present invention may exhibit a total number of up to 200 (up to 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200) changes in the amino acid sequence (i.e. exchanges,insertions, deletions, N-terminal truncations, and/or C-terminaltruncations). The amino acid residues described herein employ either thesingle letter amino acid designator or the three-letter abbreviation inkeeping with the standard polypeptide nomenclature, J. Biol. Chem.,(1969), 243, 3552-3559. All amino acid residue sequences are representedherein by formulae with left and right orientation in the conventionaldirection of amino-terminus to carboxy-terminus.

The terms “effective amount” or “therapeutically effective amount” referto that amount of a composition of the disclosure that is sufficient toeffect treatment, as defined below, when administered to a mammal inneed of such treatment. This amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the particular composition of thedisclosure chosen, the dosing regimen to be followed, timing ofadministration, manner of administration and the like, all of which canreadily be determined by one of ordinary skill in the art.

An “epitope”, also known as antigenic determinant, is the segment of amacromolecule, e.g. a protein, which is recognized by the adaptiveimmune system, such as by antibodies, B cells, or T cells. An epitope isthat part or segment of a macromolecule capable of binding to anantibody or antigen-binding fragment thereof. In this context, the term“binding” in particular relates to a specific binding. In the context ofthe present invention it is preferred that the term “epitope” refers tothe segment of protein or polyprotein that is recognized by the immunesystem.

The term galactose is well known in the art and refers to amonosaccharide sugar that exists both in open-chain form and in cyclicform, having D- and L-isomers. In the cyclic form there are two anomers,namely alpha and beta. In the alpha form, the C1 alcohol group is in theaxial position, whereas in the beta form, the C1 alcohol group is in theequatorial position. In particular, “galactose” refers to the cyclicsix-membered pyranose, more in particular the D-isomer and even moreparticularly the alpha-D-form (α-D-galactopyranose). The structure andnumbering of galactose is illustrated below.

The term “galactosylating moiety” refers to a particular type ofliver-targeting moiety. Galactosylating moieties include, but are notlimited to a galactose, galactosamine and/or N-acetylgalactosamineresidue.

The term “liver-targeting moiety”, refers to moieties having the abiityto direct, e.g., a polypeptide, to the liver. The liver comprisesdifferent cell types, including but not limited to hepatocytes,sinusoidal epithelial cells, Kupffer cells, stellate cells, and/ordendritic cells. Typically, a liver-targeting moiety directs apolypeptide to one or more of these cells. On the surface of therespective liver cells, receptors are present which recognize andspecifically bind the liver-targeting moiety. Liver-targeting can beachieved by chemical conjugation of an antigen or ligand to agalactosylating moiety, desialylation of an antigen or ligand to exposeunderlying galactosyl moieties, recombinant fusion or chemicalconjugation of an antigen or ligand to an ASGPR-binding moiety, orspecific binding of an endogenous antibody to an antigen or ligand,where the antigen or ligand is: desialylated to expose underlyinggalactosyl moieties, conjugated to a galactosylating moiety, orrecombinantly fused or chemically conjugated to an ASGPR-binding moiety.Naturally occurring desialylated proteins are not encompassed within thescope of the present disclosure.

The “numerical values” and “ranges” provided for the varioussubstituents are intended to encompass all integers within the recitedrange. For example, when defining n as an integer representing a mixtureincluding from about 1 to 100, particularly about 8 to 90 and moreparticularly about 40 to 80 ethylene glycol groups, where the mixturetypically encompasses the integer specified as n ± about 10% (or forsmaller integers from 1 to about 25, ±3), it should be understood that ncan be an integer from about 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99,100, 105 or 110) and that the disclosed mixture encompases ranges suchas 1-4, 2-4, 2-6, 3-8, 7-13, 6-14, 18-23, 26-30, 42-50, 46-57, 60-78,85-90, 90-110 and 107-113 ethylene glycol groups. The combined terms“about” and “±10%” or “±3” should be understood to disclose and providespecific support for equivalent ranges wherever used.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not.

A peptide that specifically binds a particular target is referred to asa “ligand” for that target.

A “polypeptide” is a term that refers to a chain of amino acid residues,regardless of post-translational modification (e.g., phosphorylation orglycosylation) and/or complexation with additional polypeptides, and/orsynthesis into multisubunit complexes with nucleic acids and/orcarbohydrates, or other molecules. Proteoglycans therefore also arereferred to herein as polypeptides. A long polypeptide (having overabout 50 amino acids) is referred to as a “protein.” A short polypeptide(having fewer than about 50 amino acids) is referred to as a “peptide.”Depending upon size, amino acid composition and three dimensionalstructure, certain polypeptides can be referred to as an“antigen-binding molecule,” “antibody,” an “antibody fragment” or a“ligand.” Polypeptides can be produced by a number of methods, many ofwhich are well known in the art. For example, polypeptides can beobtained by extraction (e.g., from isolated cells), by expression of arecombinant nucleic acid encoding the polypeptide, or by chemicalsynthesis. Polypeptides can be produced by, for example, recombinanttechnology, and expression vectors encoding the polypeptide introducedinto host cells (e.g., by transformation or transfection) for expressionof the encoded polypeptide

As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The term “purified” as used herein with reference to a polypeptiderefers to a polypeptide that has been chemically synthesized and is thussubstantially uncontaminated by other polypeptides, or has beenseparated or isolated from most other cellular components by which it isnaturally accompanied (e.g., other cellular proteins, polynucleotides,or cellular components). An example of a purified polypeptide is onethat is at least 70%, by dry weight, free from the proteins andnaturally occurring organic molecules with which it naturallyassociates. A preparation of a purified polypeptide therefore can be,for example, at least 80%, at least 90%, or at least 99%, by dry weight,the polypeptide. Polypeptides also can be engineered to contain a tagsequence (e.g., a polyhistidine tag, a myc tag, a FLAG® tag, or otheraffinity tag) that facilitates purification or marking (e.g., captureonto an affinity matrix, visualization under a microscope). Thus apurified composition that comprises a polypeptide refers to a purifiedpolypeptide unless otherwise indicated. The term “isolated” indicatesthat the polypeptides or nucleic acids of the disclosure are not intheir natural environment. Isolated products of the disclosure can thusbe contained in a culture supernatant, partially enriched, produced fromheterologous sources, cloned in a vector or formulated with a vehicle,etc.

The term “sequence identity” is used with regard to polypeptide sequencecomparisons. This expression in particular refers to a percentage ofsequence identity, for example at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% to the respective reference polypeptide or tothe respective reference polynucleotide. Particularly, the polypeptidein question and the reference polypeptide exhibit the indicated sequenceidentity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80,90, 100 or more amino acids or over the entire length of the referencepolypeptide.

“Specific binding,” as that term is commonly used in the biologicalarts, refers to a molecule that binds to a target with a relatively highaffinity as compared to non-target tissues, and generally involves aplurality of non-covalent interactions, such as electrostaticinteractions, van der Waals interactions, hydrogen bonding, and thelike. Specific binding interactions characterize antibody-antigenbinding, enzyme-substrate binding, and certain protein-receptorinteractions; while such molecules might bind tissues besides theirspecific targets from time to time, to the extent that such non-targetbinding is inconsequential, the high-affinity binding pair can stillfall within the definition of specific binding.

The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including:

-   -   preventing or protecting against the disease or disorder, that        is, causing the clinical symptoms not to develop;    -   inhibiting the disease or disorder, that is, arresting or        suppressing the development of clinical symptoms; and/or        relieving the disease or disorder, that is, causing the        regression of clinical symptoms.

The term “unwanted immune response” refers to a reaction by the immunesystem of a subject, which in the given situation is not desirable. Thereaction of the immune system is unwanted if such reaction does not leadto the prevention, reduction, or healing of a disease or disorder butinstead causes, enhances or worsens a disorder or disease. Typically, areaction of the immune system causes, enhances or worsens a disease ifit is directed against an inappropriate target. Exemplified, an unwantedimmune response includes but is not limited to transplant rejection,immune response against a therapeutic agent, autoimmune disease, andallergy or hypersensitivity.

The term “variant” is to be understood as a protein which differs incomparison to the protein from which it is derived by one or morechanges in its length, sequence, or structure. The polypeptide fromwhich a protein variant is derived is also known as the parentpolypeptide or polynucleotide. The term “variant” comprises “fragments”or “derivatives” of the parent molecule. Typically, “fragments” aresmaller in length or size than the parent molecule, whilst “derivatives”exhibit one or more differences in their sequence or structure incomparison to the parent molecule. Also encompassed modified moleculessuch as but not limited to post-translationally modified proteins (e.g.glycosylated, phosphorylated, ubiquitinated, palmitoylated, orproteolytically cleaved proteins) and modified nucleic acids such asmethylated DNA. Also mixtures of different molecules such as but notlimited to RNA-DNA hybrids, are encompassed by the term “variant”.Naturally occurring and artificially constructed variants are to beunderstood to be encompassed by the term “variant” as used herein.Further, the variants usable in the present invention may also bederived from homologs, orthologs, or paralogs of the parent molecule orfrom artificially constructed variant, provided that the variantexhibits at least one biological activity of the parent molecule, i.e.is functionally active. A variant can be characterized by a certaindegree of sequence identity to the parent polypeptide from which it isderived. More precisely, a protein variant in the context of the presentdisclosure may exhibit at least 80% sequence identity to its parentpolypeptide. Preferably, the sequence identity of protein variants isover a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 ormore amino acids.

Compositions

One aspect of the present disclosure relates to compositions,pharmaceutical formulations, and methods of treatment employing suchcompositions, as represented by Formula 1:

X

Y—Z]_(m)   Formula 1

where:

-   -   m is an integer from about 1 to 100, particularly from about 1        to 20, and most particularly 1 to about 10;    -   X is an antigen moiety, particularly a foreign antigen or        self-antigen against which a patient develops an unwanted immune        response, or a tolerogenic portion (e.g., a fragment or an        epitope) of such an antigen moiety;    -   Y is a linker moiety or a direct bond, or an antibody, antibody        fragment, peptide or other ligand that specifically binds X; and    -   Z is a liver-targeting moiety, in particular galactosylating        moiety.

The value for m in Formula 1 will depend upon the nature of X, in thateach antigen, antibody, antibody fragment or ligand will have anindividual number and density of sites (predominantly the N-terminalamine, lysine residues and cysteine residues) to which a linker orgalactosylating moiety can be bound. Antigens having a limited number ofsuch sites can be derivatized, for example, at the N or C terminus, byadding lysine or cysteine residues (optionally via a cleavable linker,particularly a linker having an immunoproteosome cleavage site).Generally, it is preferred to provide an adequate degree ofgalactosylation in compositions of Formula 1 so as to facilitate uptakeby liver cells. Pharmaceutical formulations and methods of thedisclosure can employ a cocktail of compositions of Formula 1,respectively bearing different X moieties (e.g., several epitopesassociated with a particular unwanted immune response).

The compositions of Formula 1 include the sub-genuses where X is aforeign transplant antigen against which transplant recipients developan unwanted immune response (e.g., transplant rejection), a foreignfood, animal, plant or environmental antigen against which patientsdevelop an unwanted immune (e.g., allergic or hypersensitivity)response, a foreign therapeutic agent against which patients develop anunwanted immune response (e.g., hypersensitivity and/or reducedtherapeutic activity), or a self-antigen against which patients developan unwanted immune response (e.g., autoimmune disease); where Y is alinker of Formulae Ya through Yp; and/or where Z is galactose,galactosamine or N-acetylgalactosamine, as illustrated by Formulae 1athrough 1p as described below with reference to the Reaction Schemes.

The disclosure further provides a pharmaceutically acceptablecomposition represented by Formula 2:

where:

-   -   m′ is zero or an integer from about 1 to 10, m″ is zero or an        integer from about 1 to 10, and the sum of m′+m″ is an integer        from about 1 to 10 but is at least 1;    -   X is an antigen moiety, particularly a foreign antigen or        self-antigen against which a patient develops an unwanted immune        response, or a tolerogenic portion (e.g., a fragment or an        epitope) of such an antigen moiety;    -   Y is a linker moiety or a direct bond, or an antibody, antibody        fragment, peptide or other ligand that specifically binds X; and    -   Z is a liver-targeting moiety, in particular an ASGPR-targeted        antibody, an ASGPR-targeted antibody fragment, an ASGPR-targeted        peptide, an ASGPR-targeted scFv, or another ASGPR ligand,        such composition optionally including amino acid sequences to        facilitate isolation and purification [e.g., a “His tag” or        “6×His” having the sequence: HHHHHH (SEQ ID NO:1) and additional        linkers [e.g., “Gly₃Ser” having the sequence: GGGS (SEQ ID        NO:2)]. In the compositions of Formula 2 where m′+m″ is greater        than one, the X moieties can be the same or different, and the Y        moieties can be the same or different. The value for m′+m″ will        be smaller (e.g., 3 or less) when X is a full protein, or larger        (up to about 10) when X is a peptide. The linker(s) “Y” can        advantageously comprise a cleavage site, particularly an        immunoproteosome cleavage site.

The compositions of Formula 2 include the sub-genuses where X is aforeign transplant antigen against which transplant recipients developan unwanted immune response (e.g., transplant rejection), a foreignfood, animal, plant or environmental antigen against which patientsdevelop an unwanted immune (e.g., allergic or hypersensitivity)response, a foreign therapeutic agent against which patients develop anunwanted immune response (e.g., hypersensitivity and/or reducedtherapeutic activity), or a self-antigen against which patients developan unwanted immune response (e.g., autoimmune disease).

Alternatively, in the compositions of Formula 1 and/or Formula 2, X canbe an antibody, antibody fragment or ligand that specifically binds acirculating protein or peptide or antibody, which circulating protein orpeptide or antibody is causatively involved in transplant rejection,immune response against a therapeutic agent, autoimmune disease,hypersensitivity and/or allergy.

The compositions of Formula 2 can be prepared as fusion proteins and caninclude several components useful in their preparation and purification,such as a signal or leader sequence that directs the expressedpolypeptide to a specific transport pathway and is subsequently cleavedfrom the polypeptide; therefore the signal sequence can be part of theexpressed protein and DNA encoding it, but not part of the finalcomposition. One example of a mammalian signal sequence used inexpression systems is the Ig κ-chain sequence, which directs proteinsecretion: METDTLLLWVLLLWVPGSTG (SEQ ID NO:3). Similarly, one example ofa common bacterial signal sequence used in expression systems is thepelB signal sequence, which directs expressed protein into the bacterialperiplasm: MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:4).

Antigens

The antigen employed as X in the compositions of Formula 1 and/orFormula 2 can be a protein or a peptide, e.g. the antigen may be acomplete or partial therapeutic agent, a full-length transplant proteinor peptide thereof, a full-length autoantigen or peptide thereof, afull-length allergen or peptide thereof, and/or a nucleic acid, or amimetic of an aforementioned antigen.

Antigens employed in the practice of the present disclosure can be oneor more of the following:

-   -   Therapeutic agents that are proteins, peptides, antibodies and        antibody-like molecules, including antibody fragments and fusion        proteins with antibodies and antibody fragments. These include        human, non-human (such as mouse) and non-natural (i.e.,        engineered) proteins, antibodies, chimeric antibodies, humanized        antibodies, and non-antibody binding scaffolds, such as        fibronectins, DARPins, knottins, and the like.    -   Human allograft transplantation antigens against which        transplant recipients develop an unwanted immune response.    -   Self-antigens that cause an unwanted, autoimmune response. Those        skilled in the art will appreciate that while self-antigens are        of an endogenous origin in an autoimmune disease patient, the        polypeptides employed in the disclosed compositions are        typically synthesized exogenously (as opposed to being purified        and concentrated from a source of origin).    -   Foreign antigens, such as food, animal, plant and environmental        antigens, against which a patient experiences an unwanted immune        response. Those skilled in the art will appreciate that while a        therapeutic protein can also be considered a foreign antigen due        to its exogenous origin, for purposes of clarity in the        description of the present disclosure such therapeutics are        described as a separate group. Similarly, a plant or an animal        antigen can be eaten and considered a food antigen, and an        environmental antigen may originate from a plant. They are,        however, all foreign antigens. In the interest of simplicity no        attempt will be made to describe distingiush and define all of        such potentially overlapping groups, as those skilled in the art        can appreciate the antigens that can be employed in the        compositions of the disclosure, particularly in light of the        detailed description and examples.        The antigen can be a complete protein, a portion of a complete        protein, a peptide, or the like, and can be derivatized (as        discussed above) for attachment to a linker and/or        galactosylating moiety, can be a variant and/or can contain        conservative substitutions, particularly maintaining sequence        identity, and/or can be desialylated.

In the embodiments where the antigen is a therapeutic protein, peptide,antibody or antibody-like molecule, specific antigens can be selectedfrom: Abatacept, Abciximab, Adalimumab, Adenosine deaminase,Ado-trastuzumab emtansine, Agalsidase alfa, Agalsidase beta, Aldeslukin,Alglucerase, Alglucosidase alfa, α-1-proteinase inhibitor, Anakinra,Anistreplase (anisoylated plasminogen streptokinase activator complex),Antithrombin III, Antithymocyte globulin, Ateplase, Bevacizumab,Bivalirudin, Botulinum toxin type A, Botulinum toxin type B, C1-esteraseinhibitor, Canakinumab, Carboxypeptidase G2 (Glucarpidase and Voraxaze),Certolizumab pegol, Cetuximab, Collagenase, Crotalidae immune Fab,Darbepoetin-α, Denosumab, Digoxin immune Fab, Dornase alfa, Eculizumab,Etanercept, Factor Vila, Factor VIII, Factor IX, Factor XI, Factor XIII,Fibrinogen, Filgrastim, Galsulfase, Golimumab, Histrelin acetate,Hyaluronidase, Idursulphase, Imiglucerase, Infliximab, Insulin[including recombinant human insulin (“rHu insulin”) and bovineinsulin], Interferon-α2a, Interferon-α2b, Interferon-β1a,Interferon-β1b, Interferon-γ1 b, Ipilimumab, L-arginase, L-asparaginase,L-methionase, Lactase, Laronidase, Lepirudin/hirudin, Mecasermin,Mecasermin rinfabate, Methoxy Natalizumab, Octreotide, Ofatumumab,Oprelvekin, Pancreatic amylase, Pancreatic lipase, Papain,Peg-asparaginase, Peg-doxorubicin HCl, PEG-epoetin-β, Pegfilgrastim,Peg-Interferon-α2a, Peg-Interferon-α2b, Pegloticase, Pegvisomant,Phenylalanine ammonia-lyase (PAL), Protein C, Rasburicase (uricase),Sacrosidase, Salmon calcitonin, Sargramostim, Streptokinase,Tenecteplase, Teriparatide, Tocilizumab (atlizumab), Trastuzumab, Type 1alpha-interferon, Ustekinumab, vW factor. The therapeutic protein can beobtained from natural sources (e.g., concentrated and purified) orsynthesized, e.g., recombinantly, and includes antibody therapeuticsthat are typically IgG monoclonal or fragments or fusions.

Particular therapeutic protein, peptide, antibody or antibody-likemolecules include Abciximab, Adalimumab, Agalsidase alfa, Agalsidasebeta, Aldeslukin, Alglucosidase alfa, Factor VIII, Factor IX,Infliximab, Insulin (including rHu Insulin), L-asparaginase, Laronidase,Natalizumab, Octreotide, Phenylalanine ammonia-lyase (PAL), orRasburicase (uricase) and generally IgG monoclonal antibodies in theirvarying formats.

Another particular group includes the hemostatic agents (Factor VIII andIX), Insulin (including rHu Insulin), and the non-human therapeuticsuricase, PAL and asparaginase.

Unwanted immune response in hematology and transplant includesautoimmune aplastic anemia, transplant rejection (generally), and Graftvs. Host Disease (bone marrow transplant rejection). In the embodimentswhere the antigen is a human allograft transplantation antigen, specificsequences can be selected from: subunits of the various MHC class I andMHC class II haplotype proteins (for example, donor/recipientdifferences identified in tissue cross-matching), and single-amino-acidpolymorphisms on minor blood group antigens including RhCE, Kell, Kidd,Duffy and Ss. Such compositions can be prepared individually for a givendonor/recipient pair.

In the embodiments where the antigen is a self-antigen, specificantigens (and the autoimmune disease with which they are associated) canbe selected from:

-   -   In type 1 diabetes mellitus, several main antigens have been        identified: insulin, proinsulin, preproinsulin, glutamic acid        decarboxylase-65 (GAD-65 or glutamate decarboxylase 2), GAD-67,        glucose-6 phosphatase 2 (IGRP or islet-specific glucose 6        phosphatase catalytic subunit related protein),        insulinoma-associated protein 2 (IA-2), and        insulinoma-associated protein 2β ((IA-2β); other antigens        include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38,        GLIMA 38, chromogranin-A, HSP-60, caboxypeptidase E, peripherin,        glucose transporter 2,        hepatocarcinoma-intestine-pancreas/pancreatic associated        protein, S100β, glial fibrillary acidic protein, regenerating        gene II, pancreatic duodenal homeobox 1, dystrophia myotonica        kinase, islet-specific glucose-6-phosphatase catalytic        subunit-related protein, and SST G-protein coupled receptors        1-5. It should be noted that insulin is an example of an antigen        that can be characterized both as a self-antigen and a        therapeutic protein antigen. For example, rHu Insulin and bovine        insulin are therapeutic protein antigens (that are the subject        of unwanted immune attack), whereas endogenous human insulin is        a self-antigen (that is the subject of an unwanted immune        attack). Because endogenous human insulin is not available to be        employed in a pharmaceutical composition a recombinant form is        employed in the compositions of the disclosure.        -   Human insulin, including an exogenously obtained form useful            in the compositions of the disclosure, has the following            sequence UNIPROT P01308):

(SEQ ID NO: 5) MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALYLVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPLALEGSLQKRG IVEQCCTSIC SLYQLENYCN.

-   -   -   GAD-65, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            UNIPROT Q05329):

(SEQ ID NO: 6) MASPGSGFWS FGSEDGSGDS ENPGTARAWC QVAQKFTGGI GNKLCALLYG DAEKPAESGG SQPPRAAARK AACACDQKPC SCSKVDVNYA FLHATDLLPA CDGERPTLAF LQDVMNILLQ YVVKSFDRST KVIDFHYPNE LLQEYNWELA DQPQNLEEIL MHCQTTLKYA IKTGHPRYFN QLSTGLDMVG LAADWLTSTANTNMFTYEIA PVFVLLEYVT LKKMREIIGW PGGSGDGIFS PGGAISNMYA MMIARFKMFP EVKEKGMAAL PRLIAFTSEH SHFSLKKGAA ALGIGTDSVI LIKCDERGKM IPSDLERRIL EAKQKGFVPF LVSATAGTTV YGAFDPLLAV ADICKKYKIW MHVDAAWGGG LLMSRKHKWK LSGVERANSV TWNPHKMMGVPLQCSALLVR EEGLMQNCNQ MHASYLFQQD KHYDLSYDTG DKALQCGRHV DVFKLWLMWR AKGTTGFEAH VDKCLELAEY LYNIIKNREG YEMVFDGKPQ HTNVCFWYIP PSLRTLEDNE ERMSRLSKVA PVIKARMMEY GTTMVSYQPL GDKVNFFRMV ISNPAATHQD IDFLIEEIER LGQDL.

-   -   -   IGRP, including an exogenously obtained form useful in the            compositions of the disclosure has the following sequence            UNIPROT QN9QR9):

(SEQ ID NO: 7) MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFFIYFPLCFQFNQTVGTKMIWVAVIGDWLNLIFKWILFGHRPYWWVQETQIYPNHSSPCLEQFPTTCETGPGSPSGHAMGASCVWYVMVTAALSHTVCGMDKFSITLHRLTWSFLWSVFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHTPGIQTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPIAKKWCANPDWIHIDTTPFAGLVRNLGVLFGLGFAINSEMFLLSCRGGNNYTLSFRLLCALTSLTILQLYHFLQIPTHEEHLFYVLSFCKSASIPLTVVAFIPYSVHMLMKQSGKKSQ.

-   -   In autoimmune diseases of the thyroid, including Hashimoto's        thyroiditis and Graves' disease, main antigens include        thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin        receptor (TSHR); other antigens include sodium iodine symporter        (NIS) and megalin. In thyroid-associated ophthalmopathy and        dermopathy, in addition to thyroid autoantigens including TSHR,        an antigen is insulin-like growth factor 1 receptor. In        hypoparathyroidism, a main antigen is calcium sensitive        receptor.    -   In Addison's Disease, main antigens include 21-hydroxylase,        17α-hydroxylase, and P450 side chain cleavage enzyme (P450scc);        other antigens include ACTH receptor, P450c21 and P450c17.    -   In premature ovarian failure, main antigens include FSH receptor        and α-enolase.    -   In autoimmune hypophysitis, or pituitary autoimmune disease,        main antigens include pituitary gland-specific protein factor        (PGSF) 1a and 2; another antigen is type 2 iodothyronine        deiodinase.    -   In multiple sclerosis, main antigens include myelin basic        protein (“MBP”), myelin oligodendrocyte glycoprotein (“MOG”) and        myelin proteolipid protein (“PLP”).        -   MBP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT P02686):

(SEQ ID NO: 8) MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNEVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR.

-   -   -   MOG, including an exogenously obtained form useful in the            compositions of the disclosure has the following sequence            UNIPROT Q16653:

(SEQ ID NO: 9) MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRN PF.

-   -   -   PLP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            UNIPROT P60201):

(SEQ ID NO: 10) MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF.

-   -   -   Peptides/epitopes useful in the compositions of the            disclosure for treating multiple sclerosis include some or            all of the following sequences, individually in a            composition of Formula 1, together in a cocktail of            compositions of Formula 1, or fused in one or more            compositions of Formula 2:

MBP13-32: (SEQ ID NO: 11) KYLATASTMDHARHGFLPRH; MBP83-99:(SEQ ID NO: 12) ENPWHFFKNIVTPRTP; MBP111-129: (SEQ ID NO: 13)LSRFSWGAEGQRPGFGYGG; MBP146-170: (SEQ ID NO: 14)AQGTLSKIFKLGGRDSRSGSPMARR; MOG1-20: (SEQ ID NO: 15)GQFRVIGPRHPIRALVGDEV; MOG35-55:  (SEQ ID NO: 16) MEVGWYRPPFSRWHLYRNGK;and PLP139-154: (SEQ ID NO: 17) HCLGKWLGHPDKFVGI.

-   -   In rheumatoid arthritis, main antigens include collagen II,        immunoglobulin binding protein, the fragment crystallizable        region of immunoglobulin G, double-stranded DNA, and the natural        and cirtullinated forms of proteins implicated in rheumatoid        arthritis pathology, including fibrin/fibrinogen, vimentin,        collagen I and II, and alpha-enolase.    -   In autoimmune gastritis, a main antigen is H+,K+-ATPase.    -   In pernicious angemis, a main antigen is intrinsic factor.    -   In celiac disease, main antigens are tissue transglutaminase and        the natural and deamidated forms of gluten or gluten-like        proteins, such as alpha-, gamma-, and omega-gliadin, glutenin,        hordein, secalin, and avenin. Those skilled in the art will        appreciate, for example, that while the main antigen of celiac        disease is alpha gliadin, alpha gliadin turns more immunogenic        in the body through deamidation by tissue glutaminase converting        alpha gliadin's glutamines to glutamic acid. Thus, while alpha        gliadin is originally a foreign food antigen, once it has been        modified in the body to become more immunogenic it can be        characterized as a self-antigen.    -   In vitiligo, a main antigen is tyrosinase, and tyrosinase        related protein 1 and 2.        -   MART1, Melanoma antigen recognized by T cells 1, Melan-A,            including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT Q16655):

(SEQ ID NO: 18) MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVP NAPPAYEKLSAEQSPPPYSP.

-   -   -   Tyrosinase, including an exogenously obtained form useful in            the compositions of the disclosure, has the following            sequence (UNIPROT P14679):

(SEQ ID NO: 19) MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRSPCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMKNGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIFEQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCRHKRKQLPEEKQPLLMEKEDYHSLYQSHL.

-   -   -   Melanocyte protein PMEL, gp100, including an exogenously            obtained form useful in the compositions of the disclosure,            has the following sequence (UNIPROT P40967):

(SEQ ID NO: 20) MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV.

-   -   In myasthenia gravis, a main antigen is acetylcholine receptor.    -   In pemphigus vulgaris and variants, main antigens are desmoglein        3, 1 and 4; other antigens include pemphaxin, desmocollins,        plakoglobin, perplakin, desmoplakins, and acetylcholine        receptor.    -   In bullous pemphigoid, main antigens include BP180 and BP230;        other antigens include plectin and laminin 5.    -   In dermatitis herpetiformis Duhring, main antigens include        endomysium and tissue transglutaminase.    -   In epidermolysis bullosa acquisita, a main antigen is collagen        VII.    -   In systemic sclerosis, main antigens include matrix        metalloproteinase 1 and 3, the collagen-specific molecular        chaperone heat-shock protein 47, fibrillin-1, and PDGF receptor;        other antigens include Scl-70, U1 RNP, Th/To, Ku, Jo1, NAG-2,        centromere proteins, topoisomerase I, nucleolar proteins, RNA        polymerase I, II and III, PM-Slc, fibrillarin, and B23.    -   In mixed connective tissue disease, a main antigen is U1snRNP.    -   In Sjogren's syndrome, the main antigens are nuclear antigens        SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose)        polymerase and topoisomerase, muscarinic receptors, and the        Fc-gamma receptor IIIb.    -   In systemic lupus erythematosus, main antigens include nuclear        proteins including the “Smith antigen,” SS-A, high mobility        group box 1 (HMGB1), nucleosomes, histone proteins and        double-stranded DNA (against which auto-antibodies are made in        the disease process).    -   In Goodpasture's syndrome, main antigens include glomerular        basement membrane proteins including collagen IV.    -   In rheumatic heart disease, a main antigen is cardiac myosin.    -   In autoimmune polyendocrine syndrome type 1 antigens include        aromatic L-amino acid decarboxylase, histidine decarboxylase,        cysteine sulfinic acid decarboxylase, tryptophan hydroxylase,        tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450        cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing        receptor protein, and the type 1 interferons interferon alpha,        beta and omega.    -   In neuromyelitis optica, a main antigen is AQP4.        -   Aquaporin-4, including an exogenously obtained form useful            in the compositions of the disclosure, has the following            sequence (UNIPROT P55087):

(SEQ ID NO: 21) MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

-   -   In uveitis, main antigens include Retinal S-antigen or        “S-arrestin” and interphotoreceptor retinold binding protein        (IRBP) or retinol-binding protein 3.        -   S-arrestin, including an exogenously obtained form useful in            the compositions of the disclosure has the following            sequence UNIPROT P10523:

(SEQ ID NO: 22) MAASGKTSKS EPNHVIFKKI SRDKSVTIYL GNRDYIDHVSQVQPVDGVVL VDPDLVKGKK VYVTLTCAFR YGQEDIDVIGLTFRRDLYFS RVQVYPPVGA ASTPTKLQES LLKKLGSNTYPFLLTFPDYL PCSVMLQPAP QDSGKSCGVD FEVKAFATDSTDAEEDKIPK KSSVRLLIRK VQHAPLEMGP QPRAEAAWQFFMSDKPLHLA VSLNKEIYFH GEPIPVTVTV TNNTEKTVKKIKAFVEQVAN VVLYSSDYYV KPVAMEEAQE KVPPNSTLTKTLTLLPLLAN NRERRGIALD GKIKHEDTNL ASSTIIKEGIDRTVLGILVS YQIKVKLTVS GFLGELTSSE VATEVPFRLMHPQPEDPAKE SYQDANLVFE EFARHNLKDA GEAEEGKRDK NDVDE.

-   -   -   IRBP, including an exogenously obtained form useful in the            compositions of the disclosure, has the following sequence            (UNIPROT P10745):

(SEQ ID NO: 23)MMREWVLLMSVLLCGLAGPTHLFQPSLVLDMAKVLLDNYCFPENLLGMQEAIQQAIKSHEILSISDPQTLASVLTAGVQSSLNDPRLVISYEPSTPEPPPQVPALTSLSEEELLAWLQRGLRHEVLEGNVGYLRVDSVPGQEVLSMMGEFLVAHVWGNLMGTSALVLDLRHCTGGQVSGIPYIISYLHPGNTILHVDTIYNRPSNTTTEIWTLPQVLGERYGADKDVVVLTSSQTRGVAEDIAHILKQMRRAIVVGERTGGGALDLRKLRIGESDFFFTVPVSRSLGPLGGGSQTWEGSGVLPCVGTPAEQALEKALAILTLRSALPGVVHCLQEVLKDYYTLVDRVPTLLQHLASMDFSTVVSEEDLVTKLNAGLQAASEDPRLLVRAIGPTETPSWPAPDAAAEDSPGVAPELPEDEAIRQALVDSVFQVSVLPGNVGYLRFDSFADASVLGVLAPYVLRQVWEPLQDTEHLIMDLRHNPGGPSSAVPLLLSYFQGPEAGPVHLFTTYDRRTNITQEHFSHMELPGPRYSTQRGVYLLTSHRTATAAEEFAFLMQSLGWATLVGEITAGNLLHTRTVPLLDTPEGSLALTVPVLTFIDNHGEAWLGGGVVPDAIVLAEEALDKAQEVLEFHQSLGALVEGTGHLLEAHYARPEVVGQTSALLRAKLAQGAYRTAVDLESLASQLTADLQEVSGDHRLLVFHSPGELVVEEAPPPPPAVPSPEELTYLIEALFKTEVLPGQLGYLRFDAMAELETVKAVGPQLVRLVWQQLVDTAALVIDLRYNPGSYSTAIPLLCSYFFEAEPRQHLYSVFDRATSKVTEVWTLPQVAGQRYGSHKDLYILMSHTSGSAAEAFAHTMQDLQRATVIGEPTAGGALSVGIYQVGSSPLYASMPTQMAMSATTGKAWDLAGVEPDITVPMSEALSIAQDIVALRAKVPTVLQTAGKLVADNYASAELGAKMATKLSGLQSRYSRVTSEVALAEILGADLQMLSGDPHLKAAHIPENAKDRIPGIVPMQIPSPEVFEELIKFSFHTNVLEDNIGYLRFDMFGDGELLTQVSRLLVEHIWKKIMHTDAMIIDMRFNIGGPTSSIPILCSYFFDEGPPVLLDKIYSRPDDSVSELWTHAQVVGERYGSKKSMVILTSSVTAGTAEEFTYIMKRLGRALVIGEVTSGGCQPPQTYHVDDTNLYLTIPTARSVGASDGSSWEGVGVTPHVVVPAEEALARAKEMLQHNQLRVKRSPGLQDH L.

In the embodiments where the antigen is a foreign antigen against whichan unwanted immune response can be developed, such as food antigens,specific antigens can be:

-   -   from peanut: conarachin (Ara h 1), allergen II (Ara h 2),        arachis agglutinin, conglutin (Ara h 6);        -   conarachin, for example has the sequence identified as            UNIPROT Q6PSU6    -   from apple: 31 kda major allergen/disease resistance protein        homolog (Mal d 2), lipid transfer protein precursor (Mal d 3),        major allergen Mal d 1.03D (Mal d 1);    -   from milk: α-lactalbumin (ALA), lactotransferrin; from kiwi:        actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like        protein (Act d 2), kiwellin (Act d 5);    -   from egg whites: ovomucoid, ovalbumin, ovotransferrin, and        lysozyme;    -   from egg yolks: livetin, apovitillin, and vosvetin;    -   from mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2),        lipid transfer protein (Sin a 3), profilin (Sin a 4);    -   from celery: profilin (Api g 4), high molecular weight        glycoprotein (Api g 5);    -   from shrimp: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m        2), tropomyosin fast isoform;    -   from wheat and/or other cerials: high molecular weight glutenin,        low molecular weight glutenin, alpha-, gamma- and omega-gliadin,        hordein, secalin and/or avenin;        -   peptides/epitopes useful in the compositions of the            disclosure for treating Celiac Disease include some or all            of the following sequences, individually in a composition of            Formula 1, together in a cocktail of compositions of Formula            1, or fused in one or more compositions of Formula 2:

DQ-2 relevant, Alpha-gliadin “33-mer” native: (SEQ ID NO: 24)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFDQ-2 relevant, Alpha-gliadin “33-mer” deamidated: (SEQ ID NO: 25)LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF DQ-8 relevant, Alpha-gliadin:(SEQ ID NO: 26) QQYPSGQGSFQPSQQNPQDQ-8 relevant, Omega-gliadin (wheat, U5UA46): (SEQ ID NO: 27)QPFPQPEQPFPW

-   -   from strawberry: major strawberry allergy Fra a 1-E (Fra a 1);        and    -   from banana: profilin (Mus xp 1).

In the embodiments where the antigen is a foreign antigen against whichan unwanted immune response is developed, such as to animal, plant andenvironmental antigens, specific antigens can, for example, be: cat,mouse, dog, horse, bee, dust, tree and goldenrod, including thefollowing proteins or peptides derived from:

-   -   weeds, (including ragweed allergens amb a 1, 2, 3, 5, and 6, and        Amb t 5; pigweed Che a 2 and 5; and other weed allergens Par j        1, 2, and 3, and Par o 1);    -   grass (including major allergens Cyn d 1, 7, and 12; Dac g 1, 2,        and 5; Hol 11.01203; Lol p 1, 2, 3, 5, and 11; Mer a 1; Pha a 1;        Poa p 1 and 5);    -   pollen from ragweed and other weeds (including curly dock, lambs        quarters, pigweed, plantain, sheep sorrel, and sagebrush), grass        (including Bermuda, Johnson, Kentucky, Orchard, Sweet vernal,        and Timothy grass), and trees (including catalpa, elm, hickory,        olive, pecan, sycamore, and walnut);    -   dust (including major allergens from species Dermatophagoides        pteronyssinus, such as Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        14, 15, 18, 20, 21, and 23; from species Dermatophagoides        farina, such as Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18,        22, and 24; from species Blomia tropicalis such as Blo t 1, 2,        3, 4, 5, 6, 10, 11, 12, 13, 19, and 21; also allergens Eur m 2        from Euroglyphus maynei, Tyr p 13 from Tyrophagus putrescentiae,        and allergens Bla g 1, 2, and 4; Per a 1, 3, and 7 from        cockroach);    -   pets (including cats, dogs, rodents, and farm animals; major cat        allergens include Fel d 1 through 8, cat IgA, BLa g 2, and cat        albumin; major dog allergens include Can f 1 through 6, and dog        albumin);    -   bee stings, including major allergens Api m 1 through 12; and    -   fungus, including allergens derived from, species of Aspergillus        and Penicillium, as well as the species Alternaria alternata,        Davidiella tassiana, and Trichophyton rubrum.

As will be appreciated by those skilled in the art, a patient can betested to identify an antigen against which an unwanted immune responsehas developed, and a protein, peptide or the like can be developed basedon that antigen and incorporated as X in a composition of the presentdisclosure.

Sialated Antigens, Antibodies, Antibody Fragments

Following are examples of antigens, antibodies, antibody fragmentshaving sialylation that can be removed to leave glycosylationspecifically targeting the ASGPR: follicle stimulating hormone (FSH),human chorionic gonadotropin (HCG), luteinizing hormone (LH),osteopontin, thyroid stimulating hormone (TSH), agalsidase alfa,agalsidase beta (FABRAZYME®; Genzyme), epoetin alfa and epoetin beta,follitropin alfa (GONAL-F®; Merck/Serono) and follitropin beta(FOLLISTIM®; Schering-Plough), insulin growth factor binding protein 6(IGFBP-6), lutropin alfa (LUVERIS®; Merck/Serono), transforming growthfactor β1, antithrombin (ATryn®/TROMBATE-III®; Genzyme/TalecrisBiotherapeutics), thyrotropin alfa (THYROGEN®; Genzyme), lenograstim,sargramostim (LEUKINE®; Genzyme), interleukin-3, prourokinase,lymphotoxin, C1-esterase inhibitor (Berinert®; CSL), IgG-likeantibodies, interferon beta, coagulation factor Vila (NOVOSEVEN®; NovoNordisk), coagulation factor VIII (moroctocog alfa), coagulation factorIX (nonacog alfa) (BENEFIX®; Wyeth), and the p55 tumor necrosis receptorfusion protein. (See: Byrne et al., Drug Discovery Today, Vol 12, No.7/8, pages 319-326, April 2007 and Sola et al., BioDrugs. 2010; 24(1):9-21). Pharmaceutically relevant proteins that have previously beenhyperglycosylated and can be desialylated for hepatocyte-ASGPRtaregeting include: interferon alfa and gamma, luteinizing hormone, Fvantibody fragments, asparaginase, cholinesterase, darbepoetin alfa(AraNESP®; Amgen), trombopoietin, leptin, FSH, IFN-α2, serum albumin,and corifollitropin alfa.

Proteins with glycans that do not normally terminate in sialic acids,including proteins produced in bacteria or yeast (such as arginase, someinsulins, and uricase) would not be amenable to desialylation.

Those skilled in the art will appreciate that publicly availablereferences, such as UNIPROT, disclose the presence and location of sitesfor desialylation on most if not all antigens, antibodies, antibodyfragments and ligands of interest.

Antibodies and Peptide Ligands

In the embodiments employing an antibody, antibody fragment or ligand,such moieties are chosen to specifically bind a targeted circulatingprotein or peptide or antibody, and result in hepatic uptake of thecirculating targeted moiety, possibly as an adduct with the targetingmoiety, ultimately resulting in the clearance and inactivation of thecirculating targeted moiety. For example, liver-targeted Factor VIIIwill bind and clear circulating Factor VIII antibodies. Procedures forthe identification of such moieties will be familiar to those skilled inthe art.

Linkers

The linkers employed in the compositions of the present disclosure (“Y”in Formula 1) can include N-hydroxysuccinamidyl linkers, malaemidelinkers, vinylsulfone linkers, pyridyl di-thiol-poly(ethylene glycol)linkers, pyridyl di-thiol linkers, n-nitrophenyl carbonate linkers,NHS-ester linkers, nitrophenoxy poly(ethylene glycol)ester linkers andthe like.

One particular group of linkers Formula Y′-CMP below (where Y′ indicatesthe remaining portion of the linker and R⁹ and Z are as defined). Moreparticularly, in the group of linkers including Formula Y′-CMP, the R⁹substituent is an ethylacetamido group, and even more particularly theethylacetamido is conjugated with C1 of N-acetylgalactosamine.

Di-thiol-containing linkers, particularly disulfanylethylcarbamate-containing linkers (named including a free amine of X,otherwise named a “disulfanyl ethyl ester” without including the freeamine of X) are particularly advantageous in the present compositions ashaving the ability to cleave and release an antigen in its original formonce inside a cell, for example as illustrated below (where Y′ indicatesthe remaining portion of the linker and X′ and Z are as defined).

Particularly with regard to the linkers illustrated below in Formula Yathrough Formula Yp:

-   -   the left bracket “(” indicates the bond between X and Y;    -   the right or bottom bracket “)” indicates the bond between Y and        Z;    -   n is an integer representing a mixture including from about 1 to        100, particularly about 8 to 90 (e.g., 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65        70, 75, 80, 85, 90 or 95), more particularly about 40 to 80        (e.g., 39, 40, 43, 45, 46, 48, 50, 52, 53, 55, 57, 60, 62, 65,        66, 68, 70, 73, 75, 78, 80 or 81) ethylene glycol groups, where        the mixture typically encompasses the integer specified as        n±10%;    -   p is an integer representing a mixture including from about 2 to        150, particularly about 20 to 100 (e.g., 18, 19, 20, 25, 30, 35,        40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 or 105) and        more particularly about 30 to 40 (e.g., 28, 29, 30, 31, 32, 33,        34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44), where the mixture        typically encompasses the integer specified as p±10%;    -   q is an integer representing a mixture including from about 1 to        44, particularly about 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22) and more        particularly about 4 to 12 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12        or 13), where the mixture typically encompasses the integer        specified as q±10%; and    -   R⁸ is —CH₂— (“methyl”) or —CH₂—CH₂—C(CH₃)(CN)—        (“1-cyano-1-methyl-propyl” or “CMP”).    -   R⁹ is a direct bond or —CH₂—CH₂—NH—C(O)— (an ethylacetamido        group or “EtAcN”), as illustrated in the following structures of        Formula 1 (where the EtAcN group is shown and the rest of the        linker is referred to as Y′):

and Z is galactose, galactosamine or N-acetylgalactosamine conjugated atC1.

(Linkers of Formula Yn can be synthesized via certain precursors thatrender Yn particularly suitable for conjugation to hydrophobicantigens.)

The linkers shown above as Formulae Yh through Yn are synthesized asisomers that are employed without separation. For example, the linkersof Formulae Yh, Yi, Yj and Yn will be a mixture of the8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8yl and8,9-dihydro-3H-dibenzo[b,][1,2,3]triazolo[4,5-d]azocin-8yl structuresillustrated below:

The linkers of Formulae Yk, YL and Ym will be a mixture of the8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8-yl and8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-9-ylstructures illustrated below:

The presence of such isomeric mixtures does not impair the functionalityof the compositions employing such linkers.

Galactosylating Moieties

The galactosylating moieties employed in the compositions of the presentdisclosure serve to target the compositions to liver cells (for example,specifically binding hepatocytes) and can be selected from: galactose,galactosamine or N-acetylgalactosamine. It has been reported that ASGPRaffinity can be retained while modifying either side of galactose'sC3/C4-diol anchor (Mamidyala, Sreeman K., et al., J. Am. Chem. Soc.2012, 134, 1978-1981), therefore the points of conjugation areparticularly at C1, C2 and C6.

Particular galactosylating moieties include galactose conjugated at C1or C6, galactosamine conjugated at C2, and N-acetylgalactosamineconjugated at C6. Other particular galactosylating moieties includeN-acetylgalactosamine conjugated at C2, more particularly conjugated toa linker bearing an R⁹ substituent that is CH₂. Still other particulargalactosylating moieties include galactose, galactosamine orN-acetylgalactosamine conjugated at C1, more particularly conjugated toa linker bearing an R⁹ substituent that is an ethylacetamido group.

ASGPR Targeting Antibodies

The ASGPR-specific antibodies employed in the compositions of thepresent disclosure also serve to target compositions of the disclosureto liver cells and can be selected from commercially available products,such as: Anti-Asialoglycoprotein Receptor 1 antibody (ab42488) fromAbcam plc, Cambridge, UK and ASGPR1/2 (FL-291) (sc28977) from Santa CruzBiotechnology, Inc., Dallas, Tex. Alternatively, such antibodies can beexpressed using any of a number of published sequences, such as theDom26h-196-61, single-domain anti-ASGPR antibody:

(SEQ.ID.No: 6) EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS HKRHEHTRFDSWGQGTLVTVSS[Coulstock E, et al., (2013) “Liver-targeting of interferon-alpha withtissue-specific domain antibodies.” PLoS One. 8(2):e57263 and US2013/0078216], or such a sequence having conservative substitutions. Theabove-referenced US patent application discloses liver-targetingmolecules such as DOM26h-196-61 for delivering certain therapeutics[including interferon (interferon alpha 2, interferon alpha 5,interferon alpha 6, or consensus interferon), Ribavirin,Nexavar/Sorafenib, Erbitus/Cetuximab, Avastatin/bevacizumab, andHerceptin/trastuzumab] for the treatment of liver diseases. Thecompositions of matter corresponding to Formula 2 employingDOM26h-196-61 or another liver-targeting molecule described in US2013/0078216 do not include interferon (interferon alpha 2, interferonalpha 5, interferon alpha 6, or consensus interferon), Ribavirin,Nexavar/Sorafenib, Erbitus/Cetuximab, Avastatin/bevacizumab, andHerceptin/trastuzumab within their scope.

New sequences for an antibody, antibody fragment, or peptide thatspecifically targets ASGPR can be discovered using various methods knownby those skilled in the art. These methods can include, but are notlimited to, vaccination technology, hybridoma technology, librarydisplay technologies (including bacterial and phage platforms),endogenous repertoire screening technologies, directed evolution andrational design.

Nomenclature

The compositions of Formula 1 can be named using a combination of IUPACand trivial names. For example, a compound corresponding to Formula 1where X is a cyclobutyl moiety (shown instead of an antigen forillustrative purposes), Y is Formula Ya, m is 1, n is 4 and Z isN-acetylgalactosamin-2-yl:

can be named(Z)-(21-cyclobutyl-1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-ylidene)triaz-1-yn-2-iumchloride, so the corresponding composition of the disclosure where X istissue transglutaminase can be named (Z)-(21-(tissuetransglutaminase)-1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-ylidene)triaz-1-yn-2-iumchloride. The corresponding composition of the disclosure where X′ istissue transglutaminase, m is 2, n is 4 and Z′ isN-acetylgalactosamin-2-yl can be named (3Z)-((tissuetransgultaminase)-1,3-diylbis(1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-yl-21-ylidene))bis(triaz-1-yn-2-ium)chloride.

In the interest of simplification, the compositions of Formula 1 can benamed using an alternative naming system by reference to X andcorrespondence to one of Formulae 1a to 1p (as illustrated in thereaction schemes) followed by recitation of the integers for variablesm, n, p and/or q, R⁸, R⁹ and identification of the galactosylatingmoiety and the position at which it is conjugated. Under this system,the composition of Formula 1a where X is ovalbumin, m is 2, n is 4 and Zis N-acetylgalactosamin-2-yl can be named “F1a-OVA-m₂-n₄-2NAcGAL.”

Similarly, the following composition of Formula 1

can be named“2-((2-(((3-(3-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbonothioyl)thio)-3,6,9,12-tetraoxa-20-azadocosyl)-3,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)carbamoyl)oxy)ethyl)disulfanyl)ethylinsulin carboxylate.” The isomer:

can be named“2-((2-(((3-(1-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbonothioyl)thio)-3,6,9,12-tetraoxa-20-azadocosyl)-1,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)carbamoyl)-oxy)ethyl)disulfanyl)ethylinsulin carboxylate” (bold lettering highlights added for convenience inidentifying the difference between the formal names). Employing thenaming system asopted for the present disclosure, both isomers can benamed “F1n-insulin-m₁-n₁-p₁-q₄-CMP-EtAcN-1 NAcGAL” where CMP indicatesthat R⁸ is 1-cyano-1-methyl-propyl, EtAcN indicates that R⁹ isethylacetamido and 1 NAcGAL indicates Z″ is N-acetylgalactosamineconjugated at C1. Absence of the abbreviation EtAcN before thedesignation for Z would indicate that R⁹ is a direct bond.

In the compositions of Formula 2, left-to-right orientation of shouldnot be taken as specifying N to C ordering absent specific indication tothe contrary. For example, the compound of Formula 2 where m′ is 1, m″is 0, X is Ovalbumin, Y is Gly₃Ser and Z is Anti-ASGPR Dom26h-196-61,can be named OVA-Gly₃Ser-DOM and read as covering both of the following:

N-OVA-Gly₃Ser-DOM-C

and

N-DOM-Gly₃Ser-OVA-C

The compositions of Formula 2, for example where m′ is 1, m″ is 0, X isOvalbumin, Y is Gly₃Ser and Z is Anti-ASGPR Dom26h-196-61 (having apurification tag attached via an additional linker) can be named asfollows:

N-OVA-Gly₃Ser-DOM-Gly₃Ser-6×His-C

or

N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C

where the C′ terminal Gly₃Ser-6×His group represents an amino acidsequence that facilitates isolation and purification.

The composition of Formula 2 where m′ is 1, m″ is 1, each X is FactorVIII, each Y is Gly₃Ser and Z is Anti-ASGPR Dom26h-196-61 (having apurification tag attached via an additional linker) can be namedFVIII-Gly₃Ser-DOM-Gly₃Ser-FVIII-Gly₃Ser-6×His and covers both of thefollowing:

N-FVIII-Gly₃Ser-DOM-Gly₃Ser-FVIII-Gly₃Ser-6×His-C

and

N-6×His-Gly₃Ser-FVIII-Gly₃Ser-DOM-Gly₃Ser-FVIII-C.

The composition of Formula 2 where m′ is 3, m″ is 0, one, the three Xantigens, respectively, are Alpha-gliadin “33-mer” deamidated (SEQ IDNO:25), Alpha-gliadin (SEQ ID NO:26) and Omega-gliadin (SEQ ID NO:27),each Y is a linker having an immunoproteosome cleavage site (“IPC”), andZ is Anti-ASGPR Dom26h-196-61 can be named:

Alpha-gliadin “33-mer”deamidated-IPC-Alpha-gliadin-IPC-Omega-gliadin-IPC-DOM.

Preparation of the Compositions of the Disclosure

The compositions of Formula 1 can be prepared, for example, by adjustingthe procedures described in Zhu, L., et al., Bioconjugate Chem. 2010,21, 2119-2127. Syntheses of certain compositions of Formula 1 are alsodescribed below with reference to Reaction Schemes 1 to 14. Othersynthetic approaches will be apparent to those skilled in the art.

Formula 101 (below) is an alternative representation of X

where R¹ is a free surface amino (—NH₂) or thiol (—SH) moiety positionedon X's three-dimensional structure so as to be accessible forconjugation to a linker, and X′ represents the remainder of X excludingthe identified free amino group(s) [(X″ is used in the reaction schemesto represent the remainder of X excluding free thiol group(s)].Depending upon the identity of X, there will be at least one (theN-terminal amine) and can be multiple R¹ groups (predominantly fromlysine residues or cysteine residues that are not involved in disulfidebonding), as represented by m, which is an integer from about 1 to 100,more typically 1 or from about 4 to 20, and most typically 1 to about10.

Variables employed in the reaction schemes are as defined above, andadditionally include the following, which should be understood to havethese meanings absent any specific indication otherwise with respect toa particular reaction scheme or step.

-   -   R² is OH or a protecting group;    -   R³ is OH, NH₂, NHAc, a protecting group or NH-protecting group;    -   R⁴ is OH or a protecting group;    -   R⁵ is OH or a protecting group;    -   R⁶ is OH or a protecting group;    -   Z′ is galactose conjugated at C1 or C6, galactosamine conjugated        at C2, or N-acetylgalactosamine conjugated at C6;    -   R⁸ is —CH₂— or —CH₂—CH₂—C(CH₃)(CN)—; and    -   R⁹ is a direct bond and Z″ is N-acetylgalactosamine conjugated        at C2;    -   R⁹ is an ethylacetamido group and Z″ is galactose, galactosamine        or N-acetylgalactosamine conjugated at C1.

Synthetic Reaction Parameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present disclosure are inertorganic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, centrifugal size exclusion chromatography,high-performance liquid chromatography, recrystallization, sublimation,fast protein liquid chromatography, gel electrophoresis, dialysis, or acombination of these procedures. Specific illustrations of suitableseparation and isolation procedures can be had by reference to theexamples hereinbelow. However, other equivalent separation or isolationprocedures can, of course, also be used.

Unless otherwise specified (including in the examples), all reactionsare conducted at standard atmospheric pressure (about 1 atmosphere) andambient (or room) temperature (about 20° C.), at about pH 7.0-8.0.

Characterization of reaction products can be made by customary means,e.g., proton and carbon NMR, mass spectrometry, size exclusionchromatography, infrared spectroscopy, gel electrophoresis.

Reaction Scheme 1 illustrates the preparation of compositions of Formula1 where Z can be galactose, galactosamine or N-acetylgalactosamine. Inthat regard and as defined above, Z′ as employed in Reaction Scheme 1encompases galactose conjugated at C1 and C6 and corresponding to thefollowing structures according to Formula 1:

galactosamine conjugated at C2 and corresponding to the followingstructure according to Formula 1:

and N-acetylgalactosamine conjugated at C6 and corresponding to thefollowing structure according to Formula 1:

As illustrated above in Reaction Scheme 1, Step 1, surface thiolgroup(s) can be generated on an antigen, antibody, antibody fragment orligand having free surface amino group(s) (Formula 101′) by contact witha Traut reagent (Formula 102) at a pH of about 8.0 for about 1 hour togive the Formula 103′, from which unreacted Traut's reagent is removed,e.g., via centrifugal size exclusion chromatography. The two structuresshown below, illustrate the product of Reaction Scheme 1, Step 1,respectively showing the free surface amino group(s) originally found onX (i.e., Formula 103‘ where X’ represents the remainder of X excludingthe identified free surface amino groups) and omitting the free surfaceamino group(s) (i.e., Formula 103). This parallels the distinctionillustrated as between X and Formula 101. The convention has beenfollowed in the subsequent reaction schemes.

In Reaction Scheme 1, Step 2, a pyridyl di-thiol-poly(ethyleneglycol)-NHS ester (Formula 104) is contacted with galactosamine (Formula105 where R³ is NH₂ and R², R⁴, R⁵ and R⁶ are OH) with stirring at aboutpH 8 for about 1 hour to give the corresponding pyridyldi-thiol-poly(ethylene glycol)-sugar of Formula 106A, which can be usedwithout further purification.

In Reaction Scheme 1, Step 3, 4,4′-dithiodipyridine (Formula 107) iscontacted with a thiol-poly(ethylene glycol)propanoic acid (Formula 108)to give the corresponding pyridyl di-thiol-poly(ethyleneglycol)propanoic acid (Formula 109).

In Reaction Scheme 1, Step 4, the acid of Formula 109 is contacted witha protected galactose or N-acetylgalactosamine of Formula 105 where R²is OH and R³, R⁴, R⁵ and R⁶ are protecting groups (“PG”), where R⁶ is OHand R², R³, R⁴ and R⁵ are PG, or where R⁶ is N-acetyl and R², R³, R⁴ andR⁵ are PG to give the corresponding pyridyl di-thiol-poly(ethyleneglycol)-sugars of Formulae 106B, 106C and 106D, which can be usedfollowing de-protection.

In Reaction Scheme 1, Step 5, to a stirred solution of the product ofStep 1 (Formula 103′) is added an excess (corresponding to the value ofm) of the product of Step 2 or Step 4 (Formula 106, i.e., 106A, 106B,106C or 106D) for about 1 hour, followed by centrifugal sized exclusionchromatography to remove any free remaining reactants to yield thecorresponding product according to Formula 1a, respectively, Formula1aA, Formula 1aB, Formula 1aC and Formula 1aD.

The compositions corresponding to Formula 1a can be named, respectively,e.g., as follows:

“F1aA-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-2NGAL”

“F1aB-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-1GAL”

“F1aC-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-6GAL”

“F1aD-X′-m_(m)-n_(n)” or “F1a-X′-m_(m)-n_(n)-6NAcGAL”

respectively, for products made employing an intermediate according toFormulae 106A-D.

Turning to Reaction Schemes 2-14 and for the purposes of thenomenclature employed therewith, except as expressly stated otherwise,Z″ refers to N-acetylgalactosamine conjugated at C2:

or to galactose, galactosamine or N-acetylgalactosamine conjugated atC1. It should be noted that the C1 conjugated compositions need to beprotected during synthesis, for example by cyclizing the amine with theC3 hydroxyl and de-protecting following incorporation of the protectedgalactosamine into the adjacent portion of the linker.

The poly(galactose methacrylate) reactants of Formulae 201, 401, 501,601, 701, 803 and 1401 can be prepared by methacrylating galactose,e.g., contacting galactosamine and methacrylate anhydride, followed byreversible addition-fragmentation chain transfer (RAFT) polymerizationwith a corresponding RAFT agent in the presence ofazobisisobutyronitrile (AIBN) in a suitable solvent, starting withfreeze-thaw cycles followed by heating at about 60-80° C., preferably70° C. for about 5-8, preferably about 6 hours. The polymer can beprecipitated in a lower alkanol, preferably methanol.

As illustrated in Reaction Scheme 2, an antigen, antibody, antibodyfragment or ligand having free surface thiol group(s) prepared, e.g., asdescribed with reference to Reaction Scheme 1, Step 1 (Formula 103′) iscontacted with an excess (corresponding to the value of m) of a pyridyldi-thiol-poly(ethylene glycol) of Formula 201 for about 1 hour to yieldthe corresponding product according to Formula 1b.

The compositions of Formula 1b can be named as follows:

“F1b-X′-m_(m)-n_(n)-p_(p)-2NAcGAL” or“F1b-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

For example, the composition of Formula 1 b where X′ is uricase, m is 1,n is 4, p is 4 and Z″ is N-acetylgalactosamine conjugated at C2 can benamed “F1b-uricase-m₁-n₄-p₄-2NAcGAL” or“30-(uricase)-3,5,7,9-tetramethyl-12-oxo-1-phenyl-1-thioxo-3,5,7,9-tetrakis((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)carbamoyl)-13,16,19,22-tetraoxa-2,25,26-trithiatriacontan-30-iminium”.

As illustrated in Reaction Scheme 3, an antigen, antibody, antibodyfragment or ligand having native free surface thiol group(s) (cysteines)[Formula 101″ corresponding to Formula 101 and illustrating where X″, asthe term will be subsequently employed, represents X excluding theidentified free surface thiol group(s)] is contacted with an excess(corresponding to the value of m) of a pyridyl di-thiol-poly(ethyleneglycol) of Formula 201 to yield the corresponding product according toFormula 1c.

The compositions corresponding to Formula 1c can be named as follows:

“F1c-X′-m_(m)-n_(n)-p_(p)-2NAcGAL” or“F1c-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 4, an antigen, antibody, antibodyfragment or ligand having native free surface thiol group(s) of Formula101″ is contacted with an excess (corresponding to the value of m) of apyridyl di-thiol of Formula 401 to yield the corresponding productaccording to Formula 1 d.

The compositions corresponding to Formula 1d can be named as follows:

“F1d-X′-m_(m)-p_(p)-2NAcGAL” or “F1d-X′-m_(m)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 5, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) of Formula101′ is contacted with an excess (corresponding to the value of m) of an-nitrophenyl carbonate of Formula 501 to yield the correspondingproduct according to Formula 1e.

The compositions corresponding to Formula 1e can be named as follows:

“F1e-X′-m_(m)-p_(p)-2NAcGAL” or “F1e-X′-m_(m)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 6, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) of Formula101′ is contacted with an excess (corresponding to the value of m) of an-nitrophenyl carbonate poly(ethylene glycol)ester of Formula 601 toyield the corresponding product according to Formula 1f.

The compositions corresponding to Formula 1f can be named as follows:

“F1f-X′-m_(m)-n_(n)-p_(p)-2NAcGAL” or“F1f-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

As illustrated in Reaction Scheme 7, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) of Formula101′ is contacted with an excess (corresponding to the value of m) of aNHS-ester poly(ethylene glycol)ester of Formula 701 to yield thecorresponding product according to Formula 1g.

The compositions corresponding to Formula 1g can be named as follows:

“F1g-X′-m_(m)-p_(p)-2NAcGAL” or “F1g-X′-m_(m)-p_(p)-EtAcN-Z”

As illustrated in Reaction Scheme 8, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is contacted with an excess (corresponding to the value ofm) of an amine-reactive linker for Click chemistry of Formula 801 toyield the corresponding product according to Formula 802.

In Reaction Scheme 8, Step 2, the product of Formula 802 is thencontacted with an equivalent amount (again corresponding to the value ofm) of a galactos(amine) polymer of Formula 803 to yield thecorresponding isomeric product according to Formula 1 h. The twoisomers, illustrated above, result from non-specific cyclization of theazide of Formula 803 with the triple bond of Formula 802. Suchnon-specific cyclization occurs in the synthesis of other compositionswhere Y is selected from Formulae Yh through Yn, but will not beillustrated in each instance.

The compositions corresponding to Formula 1 h can be named as follows:

“F1h-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL” or“F1h-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

As illustrated in Reaction Scheme 9, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface thiol group(s) ofFormula 101″ is contacted with an excess (corresponding to the value ofm) of a thiol-reactive linker for Click chemistry of Formula 901 toyield the corresponding product according to Formula 902″.

In Reaction Scheme 9, Step 2, the product of Formula 902″ is thencontacted with an equivalent amount (again corresponding to the value ofm) of a galactos(amine) polymer of Formula 803 to yield thecorresponding isomeric product according to Formula 1i.

The compositions corresponding to Formula 1i can be named as follows:

“F1i-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL” or“F1i-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

By following the procedures described with regard to Reaction Scheme 9,but substituting starting material 101″ with a compound of Formula 103′(derivatized with the Traut reagent) there is obtained the correspondingisomeric product of Formula 1j as shown below.

The compositions corresponding to Formula 1j can be named as follows:

“F1j-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL” or“F1j-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

As illustrated in Reaction Scheme 10, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface thiol group(s) ofFormula 101″ is contacted with an excess (corresponding to the value ofm) of a thiol-reactive linker for Click chemistry of Formula 1001 toyield the corresponding product according to Formula 1002.

In Reaction Scheme 10, Step 2, the product of Formula 1002 is thencontacted with an equivalent amount (again corresponding to the value ofm) of a galactos(amine) polymer of Formula 803 to yield thecorresponding isomeric product according to Formula 1k.

The compositions corresponding to Formula 1k can be named as follows:

“F1k-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL” or“F1k-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

By following the procedures described with regard to Reaction Scheme 10,but substituting starting material 101″ with a compound of Formula 103′(derivatized with the Traut reagent) there is obtained the correspondingisomeric product of Formula 1L as shown below.

The compositions corresponding to Formula 1L can be named as follows:

“F1L-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL” or“F1L-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z”.

As illustrated in Reaction Scheme 11, Step 1, galactose, protectedgalactosamine or N-Acetyl-D-galactosamine (Formula 1101 where R³ and R⁴are OH, R³ is NH-protecting group (e.g., cyclized with R⁴) or R³ is NHAcand R⁴ is OH, respectively) is contacted with 2-chloroethan-1-olfollowed by cooling and the dropwise addition of acetylchloride. Thesolution is warmed to room temperature and then heated to 70° C. forseveral hours. Ethanol is added to the crude product and the resultingsolution is stirred in the presence of carbon and then filtered followedby solvent removal to yield the corresponding product of Formula 1102.

As illustrated in Reaction Scheme 11, Step 2, the product of Formula1102 is added to an excess of sodium azide and heated to 90° C. forseveral hours, then filtered followed by solvent removal to yield thecorresponding product of Formula 1103.

As illustrated in Reaction Scheme 11, Step 3, the product of Formula1103 is added to a solution of palladium on carbon and ethanol, andstirred under hydrogen gas (3 atm) for several hours, then filteredfollowed by solvent removal to yield the corresponding product ofFormula 1104.

As illustrated in Reaction Scheme 11, Step 4, the product of Formula1104 is added to a solution of methacrylate anhydride. Triethylamine isadded and the reaction stirred for 2 hours followed by solvent removaland isolation to yield the corresponding product of Formula 1105.

As illustrated in Reaction Scheme 11, Step 5, an azide-modified uRAFTagent (Formula 1106) is added to a solution of the product of Formula1105 with azobisisobutyronitrile, subjected to 4 free-pump-thaw cyclesand then stirred at 70° C. After several hours the corresponding polymerproduct of Formula 1107 is precipitated by addition of a lower alkanolfollowed by solvent removal. Where R³ is NH-protecting group (e.g.,cyclized with R⁴) the protecting group(s) is(are) removed at this point.

As illustrated in Reaction Scheme 11, Step 6, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is added to a pH 8.0 buffer and contacted with an excess(corresponding to the value of m) of a dioxopyrrolidine of Formula 1108with stirring. After 1 hour unreacted Formula 1108 is removed and theresulting product of Formula 1109 is used without further purification.

As illustrated in Reaction Scheme 11, Step 7, the product of Formula1107 is added to a pH 8.0 buffer, to which is added the product ofFormula 1109. After stirring for 2 hours, the excess Formula 1107 isremoved to yield the corresponding isomeric product of Formula 1 m.

By substitutingN-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)methacrylamidefor the product of Formula 1105 in Step 5 and continuing with Steps 6and 7, the corresponding isomeric product of Formula 1 m where Z″ isN-acetylgalactosamine conjugated at C2 are obtained.

The compositions corresponding to Formula 1 m can be named as follows:

“F1m-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z” where Z″ is 1 GAL, 1 NGAL or 1NAcGAL, or “F1m-X′-m_(m)-n_(n)-p_(p)-q_(q)-2NAcGAL”.

The synthetic approach of Reaction Scheme 12 is particularly suitablefor hydrophobic antigens, antibodies, antibody fragments and ligands(e.g., Insulin) due to the use of organic solvents.

As illustrated in Reaction Scheme 12, Step 1, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is dissolved in an organic solvent (e.g., DMF) containingtriethylamine. To this is added an amount (corresponding to the value ofm) of a compound of Formula 1201 followed by stirring and the additionof t-butyl methyl ether. The corresponding product of Formula 1202 isrecovered as a precipitate.

The product of Formula 1202 is resuspended in the organic solvent and anamount (corresponding to the value of m) of Formula 1107 (obtained,e.g., as described with reference to Reaction Scheme 11) is addedfollowed by stirring. The reaction product is precipitated via theaddition of dichloromethane, followed by filtration and solvent removal.Purification (e.g., resuspension in PBS followed by centrifugal sizeexclusion chromatography yields the corresponding isomeric product ofFormula 1n.

The compositions corresponding to Formula 1n can be named as follows:

“F1n-X′-m_(m)-n_(n)-p_(p)-q_(q)-EtAcN-Z” where Z″ is 1 GAL, 1 NGAL or 1NAcGAL, or “F1m-X′-nm-n_(n)-p_(p)-q_(q)-2NAcGAL”.

In Reaction Scheme 13, Step 1, a nitrophenoxycarbonyl-oxyalkyldi-thiol-poly(ethylene glycol)-NHS ester (Formula 1301) is contactedwith galactose, galactosamine or N-acetylgalactosamine (Formula 105) togive the corresponding product of Formula 1302, along with the other twoillustrated products, from which the desired nitrophenoxycarbonyldi-thiol-poly(ethylene glycol)-carboxyethyl galactose, galactosamine orN-acetylgalactosamine of Formula 1302 is isolated before proceeding tothe next step.

As illustrated in Reaction Scheme 13, Step 2, an antigen, antibody,antibody fragment or ligand having native free surface amino group(s) ofFormula 101′ is contacted with an excess (corresponding to the value ofm) of the product of Formula 1302 to yield the corresponding productaccording to Formula 10.

The compositions corresponding to Formula 10 can be named as follows:

“F10-X′-m_(m)-n_(n)-Z′.”

As illustrated in Reaction Scheme 14, an antigen, antibody, antibodyfragment or ligand having native free surface amino group(s) (Formula101′) is contacted with an excess (corresponding to the value of m) of apyridyl di-thiol-poly(ethylene glycol)-NHS ester of Formula 1401 toyield the corresponding product according to Formula 1p.

The compositions corresponding to Formula 1p can be named as follows:

“F1p-X′-m_(m)-n_(n)-p_(p)-2NAcGAL” or“F1p-X′-m_(m)-n_(n)-p_(p)-EtAcN-Z”.

Preparation of Fusion Proteins

Fusion protein compositions of Formula 2 can be expressed viaart-accepted methodology using commercially available mammalian,bacterial, yeast, or insect cell expression vectors, and published,discovered, or engineered gene sequences. Sequences encoding X, Y and Ztogether with tag sequences can be cloned into an expression vector, forexample, into the mammalian expression vector pSecTag A, where thefusion protein is inserted C-terminal to the Ig κ-chain secretion leadersequence. Various other cloning techniques can be employed, includingsite-directed mutagenesis and variations of the QuikChange protocol(Geiser, et al.), and are known by those skilled in the art. Fusionproteins can be transiently expressed in mammalian cells [e.g., in humanembryonic kidney (HEK293) cells or Chinese hamster ovary (CHO) cells] bytransient transfection with the above-described vectors usingpolyethylenimine. Transfected cells are cultured in a suitable medium(e.g., FreeStyle 293 medium, Life Technologies) supplemented, forexample, with valproic acid or DMSO for about 7 days, after which thecells are removed by centrifugation and the culture supernatants arecollected and sterilized by filtration.

Alternatively, the fusion proteins can be stably expressed by creatingstably transfected mammalian cell lines. Additionally, expressionvectors can be used to produce fusion proteins in bacteria, such asEscherichia coli, Corynebacterium, or Pseudomonas fluorescens by usingcompatible media (e.g LB, 2XYT, SOB, SOC, TB and other broths),supplements (e.g. glycerol, glucose, and other supplements), andappropriate growth and expression conditions. Expression systems inyeast can commonly use Saccharomyces cerevisiae and Pichia pastoris, orother organisms for the genera Saccharomyces, Pichia, Kluyveromyces, andYarrowia, and less commonly used organisms like the filamentous fungiAspergillus, Trichoderma, or Myceliophthora thermophila C1. Insectexpression systems can utilize baculovirus infected insect cells ornon-lytic insect cell expression to achieve protein expression levels inhigh quantity; most common insect cells include but are not limited toSf9 and Sf21 (from Spodoptera frugiperda), Hi-5 (from Trichoplusia ni),and Schneider 2 and 3 (from Drosophila melanogaster).

The expressed fusion protein products can be purified from the culturesupernatants by affinity chromatography (e.g., using a HisTrap Ni²⁺sepharose column, GE Healthcare, for a His-tagged fusion protein),followed by other chromatographic polishing steps such as size exclusionchromatography (e.g., using a Superdex 75 column, GE Healthcare) or ionexchange chromatography. Protein purity can be verified, e.g., byCoomassie Brilliant Blue staining of SDS-PAGE gels and western blotting(e.g. anti-6×His tag western blotting for a His-tagged fusion protein).Protein concentration can be determined using the Beer-Lambert Law, forwhich the absorbance at 280 nm can be measured, e.g., using a NanoDrop2000 (Thermo Scientific). The molecular weight and extinctioncoefficient can be estimated from the protein's amino acid sequence,e.g., using the ExPASy ProtParam tool. Endotoxin levels can be measured,e.g., using the HEK-Blue TLR4 reporter cell line (Invivogen) accordingto manufacturer's instructions.

Preparation of Desialylated Antigens, Antibodies, Antibody Fragments andLigands

Desialylated proteins can be produced via art-accepted methodology usingcommercially available neuraminidase (also known as acetyl-neuraminylhydrolase or sialidase) enzyme or sulfuric acid. For enzymaticdesialylation of a protein of interest, the protein can be incubatedtogether with neuraminidase at 37° C. for 1 hour, or longer asnecessary. For chemical desialylation through acid hydrolysis, a proteinof interest can be treated with 0.025 N sulfuric acid at 80° C. for 1hour, or longer as necessary. The desialylated protein can then bepurified from the reaction mixture by immobilized metal ion affinitychromatography (e.g., using a HisTrap Ni2+ sepharose column, GEHealthcare), followed by size exclusion chromatography (e.g., using aSuperdex 75 column, GE Healthcare). Protein purity can be verified,e.g., by Coomassie Brilliant Blue staining of SDS-PAGE gels andanti-6×His tag western blotting. Desialylation can be verified, e.g. bylectin-based detection of protein sialic acid content in western blotsor colorimetric quantification of sialic acid content using commerciallyavailable kits (e.g. Abcam, ProZyme, or Sigma). Desialylated proteinconcentration can be determined using the Beer-Lambert Law, for whichthe absorbance at 280 nm can be measured, e.g., using a NanoDrop 2000(Thermo Scientific). The molecular weight and extinction coefficient canbe estimated from the protein's amino acid sequence, e.g., using theExPASy ProtParam tool. Endotoxin levels can be measured, e.g., using theHEK-Blue TLR4 reporter cell line (Invivogen) according to manufacturer'sinstructions.

Particular Processes and Last Steps

A compound of Formula 103′ is contacted with an excess (corresponding tothe value of m) of a compound of Formula 106 to give the correspondingproduct of Formula 1 a.

A compound of Formula 103′ is contacted with an excess (corresponding tothe value of m) of a compound of Formula 201 to give the correspondingproduct of Formula 1b.

A compound of Formula 802, 902 or 1002 is contacted with an excess(corresponding to the value of m) of a compound of Formula 803 to givethe corresponding product of Formula 1 h, Formula 1i or Formula 1k,respectively.

A compound of Formula 1109 is contacted with an excess (corresponding tothe value of m) of a compound of Formula 1107 to give the correspondingproduct of Formula 1 m, particularly where n is about 80, p is about 30,q is about 4, and m being a function of the antigen is about 2 to 10.

A compound of Formula 1202 is contacted with an excess (corresponding tothe value of m) of a compound of Formula 1107 to give the correspondingproduct of Formula 1 n, particularly where n is about 1, p is about 30,q is about 4, and m being a function of the antigen is about 2 to 10.

Particular Compositions

By way of non-limiting example, a particular group preferred for thecompositions, pharmaceutical formulations, methods of manufacture anduse of the present disclosure are the following combinations andpermutations of substituent groups of Formula 1 (sub-grouped,respectively, in increasing order of preference):

-   -   X is a foreign transplant antigen against which transplant        recipients develop an unwanted immune response, a foreign        antigen to which patients develop an unwanted immune response, a        therapeutic protein to which patients develop an unwanted immune        response, a self-antigen to which patients develop an unwanted        immune response, or a tolerogenic portion thereof.    -   X is a therapeutic protein to which patients develop an unwanted        immune response selected from: Abatacept, Abciximab, Adalimumab,        Adenosine deaminase, Ado-trastuzumab emtansine, Agalsidase alfa,        Agalsidase beta, Aldeslukin, Alglucerase, Alglucosidase alfa,        α-1-proteinase inhibitor, Anakinra, Anistreplase (anisoylated        plasminogen streptokinase activator complex), Antithrombin III,        Antithymocyte globulin, Ateplase, Bevacizumab, Bivalirudin,        Botulinum toxin type A, Botulinum toxin type B, C1-esterase        inhibitor, Canakinumab, Carboxypeptidase G2 (Glucarpidase and        Voraxaze), Certolizumab pegol, Cetuximab, Collagenase,        Crotalidae immune Fab, Darbepoetin-α, Denosumab, Digoxin immune        Fab, Dornase alfa, Eculizumab, Etanercept, Factor Vila, Factor        VIII, Factor IX, Factor XI, Factor XIII, Fibrinogen, Filgrastim,        Galsulfase, Golimumab, Histrelin acetate, Hyaluronidase,        Idursulphase, Imiglucerase, Infliximab, Insulin (including rHu        insulin and bovine insulin), Interferon-α2a, Interferon-α2b,        Interferon-β1a, Interferon-β1b, Interferon-γ1b, Ipilimumab,        L-arginase, L-asparaginase, L-methionase, Lactase, Laronidase,        Lepirudin/hirudin, Mecasermin, Mecasermin rinfabate, Methoxy        Ofatumumab, Natalizumab, Octreotide, Oprelvekin, Pancreatic        amylase, Pancreatic lipase, Papain, Peg-asparaginase,        Peg-doxorubicin HCl, PEG-epoetin-β, Pegfilgrastim,        Peg-Interferon-α2a, Peg-Interferon-α2b, Pegloticase,        Pegvisomant, Phenylalanine ammonia-lyase (PAL), Protein C,        Rasburicase (uricase), Sacrosidase, Salmon calcitonin,        Sargramostim, Streptokinase, Tenecteplase, Teriparatide,        Tocilizumab (atlizumab), Trastuzumab, Type 1 alpha-interferon,        Ustekinumab, and vW factor.        -   Especially where X is Abciximab, Adalimumab, Agalsidase            alfa, Agalsidase beta, Aldeslukin, Alglucosidase alfa,            Factor VIII, Factor IX, Infliximab, L-asparaginase,            Laronidase, Natalizumab, Octreotide, Phenylalanine            ammonia-lyase (PAL), or Rasburicase (uricase).            -   Particularly where X is Factor VIII, Factor IX, uricase,                PAL or asparaginase.    -   X is a self-antigen polypeptide selected for treating type 1        diabetes mellitus, pediatric multiple sclerosis, juvenile        rheumatoid arthritis, celiac disease, or alopecia universalis.        -   Especially where X is a self-antigen polypeptide selected            for treating new onset type 1 diabetes mellitus, pediatric            multiple sclerosis or celiac disease.    -   X is a foreign antigen to which patients develop an unwanted        immune response        -   From peanut, including conarachin (Ara h 1)        -   From wheat, including Alpha-gliadin “33-mer” native (SEQ ID            NO:24), Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25),            Alpha-gliadin (SEQ ID NO:26) and Omega-gliadin (SEQ ID            NO:27).        -   From cat, including Fel d 1A (UNIPROT P30438) and Cat            albumin (UNIPROT P49064).        -   From dog, including Can f 1 (UNIPROT 018873) and Dog albumin            (UNIPROT P49822).    -   X is a foreign transplant antigen against which transplant        recipients develop an unwanted immune response, e.g. a human        leukocyte antigen protein.    -   X is an antibody, antibody fragment or ligand that specifically        binds a circulating protein or peptide or antibody, which        circulating protein or peptide or antibody gives rise to        transplant rejection, immune response against a therapeutic        agent, autoimmune disease, and/or allergy.        -   Especially where X binds an endogenous circulating protein            or peptide or antibody.    -   Y is a linker selected from: Formula Ya, Formula Yb, Formula Yh,        Formula Yi, Formula Yk, Formula Ym, Formula Yn, Formula Yo and        Formula Yp.        -   Especially where n is 8 to 90±10%, p is 20 to 100±10%, and q            is 3 to 20±3.    -   Particularly where n is 40 to 80±10%, p is 30 to 40±10%, and q        is 4 to 12±3.        -   Especially where Y is Formula Ya, Formula Yb, Formula Ym or            Formula Yn.            -   Particularly where n is 8 to 90±10%, p is 20 to 100±10%                and q is 3 to 20±3.                -   More particularly where n is 40 to 80±10%, p is 30                    to 40±10%, and q is 4 to 12±3.            -   Particularly where Z is conjugated to Y via an                ethylacetamido group.                -   More particularly where Z is conjugated to Y at its                    C1.                -    More particularly where R⁸ is CMP.                -   More particularly where R⁸ is CMP.            -   Particularly where R⁸ is CMP.    -   Z is galactose, galactosamine or N-acetylgalactosamine.        -   Especially where Z is galactose or N-acetylgalactosamine            conjugated at C1, C2 or C6.            -   Particularly where Z is galactose or                N-acetylgalactosamine conjugated at C1 or C2.                -   More particularly where Z is N-acetylgalactosamine                    conjugated at C1.

Each of the above-described groups and sub-groups are individuallypreferred and can be combined to describe further preferred aspects ofthe disclosure, for example but not by way of limitation, as follows:

-   -   X is a self-antigen polypeptide selected for treating type 1        diabetes mellitus, pediatric multiple sclerosis, juvenile        rheumatoid arthritis, celiac disease, or alopecia universalis.        -   Especially where X is a self-antigen polypeptide selected            for treating new onset type 1 diabetes mellitus, pediatric            multiple sclerosis or celiac disease.            -   Particularly where Y is a linker selected from: Formula                Ya, Formula Yb, Formula Yh, Formula Yi, Formula Yk,                Formula Ym, Formula Yn, Formula Yo and Formula Yp.                -   Especially where n is 8 to 90±10%, p is 20 to                    100±10%, and q is 3 to 20±3.                -    Particularly where n is 40 to 80±10%, p is 30 to                    40±10%, and q is 4 to 12±3.                -   Especially where Y is Formula Ya, Formula Yb,                    Formula Ym or Formula Yn.                -    Particularly where n is 8 to 90±10%, p is 20 to                    100±10% and q is 3 to 20±3.                -   °More particularly where n is 40 to 80±10%, p is 30                    to 40±10%, and q is 4 to 12±3.                -   +Even more particularly where Z is conjugated to Y                    via an ethylacetamido group.                -   °More particularly where Z is conjugated to Y via an                    ethylacetamido group.                -    Particularly where Z is conjugated to Y via an                    ethylacetamido group.                -   Especially where Z is galactose, galactosamine or                    N-acetylgalactosamine.                -    Particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1, C2 or C6.                -   °More particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1 or C2.                -   +Even more particularly where Z is                    N-acetylgalactosamine conjugated at C1.            -   Particularly where Z is galactose, galactosamine or                N-acetylgalactosamine.                -   Especially where Z is galactose or                    N-acetylgalactosamine conjugated at C1, C2 or C6.                -    Particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1 or C2.                -   °More particularly where Z is N-acetylgalactosamine                    conjugated at C1.        -   Especially where Y is a linker selected from: Formula Ya,            Formula Yb, Formula Yh, Formula Yi, Formula Yk, Formula Ym,            Formula Yn, Formula Yo and Formula Yp.            -   Paritcularly where n is 8 to 90±10%, p is 20 to 100±10%,                and q is 3 to 20±3.                -   More particularly where n is 40 to 80±10%, p is 30                    to 40±10%, and q is 4 to 12±3.            -   Particularly where Y is Formula Ya, Formula Yb, Formula                Ym or Formula Yn.                -   More particularly where n is 8 to 90±10%, p is 20 to                    100±10% and q is 3 to 20±3.                -    More preferably where n is 40 to 80±10%, p is 30 to                    40±10%, and q is 4 to 12±3.                -   More particularly where Z is conjugated to Y via an                    ethylacetamido group.        -   Especially where Z is galactose, galactosamine or            N-acetylgalactosamine.            -   Particularly where Z is galactose or                N-acetylgalactosamine conjugated at C1, C2 or C6.                -   More particularly where Z is galactose or                    N-acetylgalactosamine conjugated at C1 or C2.                -    More preferably where Z is N-acetylgalactosamine                    conjugated at C1.    -   m is an integer from about 1 to 100.        -   m is 1, 2, 3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16,            17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75,            80, 85, 90, 95, 100 or 110.        -   Particularly m is from about 1 to 20.            -   m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,                16, 17, 18, 19, 20, 21 or 22.            -   More particularly m is about 10.                -   m is 9, 10 or 11.    -   n is an integer representing a mixture including from about 1 to        100        -   n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,            17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54,            55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100,            105 or 110.            -   Particularly n is about 8 to 90.            -   Particularly n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,                18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54,                55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95 or                99.                -   More particularly n is about 40 to 80.                -   More particularly n is 37, 40, 41, 45, 50, 54, 55,                    59, 60, 65, 70, 75, 80, 82, 83 or 88.        -   n represents a mixture encompassing the ranges 1-4, 2-4,            2-6, 3-8, 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82,            85-90, 90-110 and 107-113.            -   Particularly n represents a mixture encompasing the                ranges 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82,                85-90 and 82-99.                -   More particularly n represents a mixture encompasing                    the ranges 36-44, 42-50, 46-57, 60-82 and 75-85.        -   p is an integer representing a mixture including from about            2 to 150.            -   p is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,                17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70,                75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 or                165.            -   Particularly where n is an integer representing a                mixture including from about 1 to 100.                -   Particularly n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,                    12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34,                    35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75,                    80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110.                -    More particularly where n is about 8 to 90.                -    More particularly n is 8, 9, 10, 11, 12, 13, 14,                    15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,                    41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83,                    85, 88, 90, 95 or 99.                -   °Even more particularly where n is about 40 to 80.                -   °Even more particularly n is 37, 40, 41, 45, 50, 54,                    55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.                -   More particularly p is 18, 19, 20, 25, 30, 35, 40,                    45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 or                    110.                -    Particularly where n is an integer representing a                    mixture including from about 1 to 100.                -   °Particularly n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,                    11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30,                    34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70,                    75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110.                -   +More particularly where n is about 8 to 90.                -   +More particularly n is 8, 9, 10, 11, 12, 13, 14,                    15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,                    41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83,                    85, 88, 90, 95 or 99.                -   ′Even more particularly where n is about 40 to 80.                -   ′Even more particularly n is 37, 40, 41, 45, 50, 54,                    55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.                -   °More particularly p is 27, 28, 29, 30, 31, 32, 33,                    34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.                -   +Particularly where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,                    10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25,                    30, 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65,                    70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or                    110.                -   ′More particularly where n is about 8 to 90.                -   ′More particularly n is 8, 9, 10, 11, 12, 13, 14,                    15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40,                    41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83,                    85, 88, 90, 95 or 99.                -   ═Even more particularly where n is about 40 to 80.                -   °Even more particularly n is 37, 40, 41, 45, 50, 54,                    55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.        -   q is an integer representing a mixture including from about            1 to 44.            -   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,                16, 17, 18, 19, 20, 25, 30, 35, 40, 44 or 48.

By way of non-limiting example, a particular group preferred for thecompositions, pharmaceutical formulations, methods of manufacture anduse of the present disclosure are the following combinations andpermutations of substituent groups of Formula 2 (sub-grouped,respectively, in increasing order of preference):

-   -   X is a therapeutic protein to which patients develop an unwanted        immune response selected from: Abatacept, Abciximab, Adalimumab,        Adenosine deaminase, Ado-trastuzumab emtansine, Agalsidase alfa,        Agalsidase beta, Aldeslukin, Alglucerase, Alglucosidase alfa,        α-1-proteinase inhibitor, Anakinra, Anistreplase (anisoylated        plasminogen streptokinase activator complex), Antithrombin III,        Antithymocyte globulin, Ateplase, Bevacizumab, Bivalirudin,        Botulinum toxin type A, Botulinum toxin type B, C1-esterase        inhibitor, Canakinumab, Carboxypeptidase G2 (Glucarpidase and        Voraxaze), Certolizumab pegol, Cetuximab, Collagenase,        Crotalidae immune Fab, Darbepoetin-α, Denosumab, Digoxin immune        Fab, Dornase alfa, Eculizumab, Etanercept, Factor Vila, Factor        VIII, Factor IX, Factor XI, Factor XIII, Fibrinogen, Filgrastim,        Galsulfase, Golimumab, Histrelin acetate, Hyaluronidase,        Idursulphase, Imiglucerase, Infliximab, Insulin, Interferon-α2a,        Interferon-α2b, Interferon-β1a, Interferon-β1b, Interferon-γ1b,        Ipilimumab, L-arginase, L-asparaginase, L-methionase, Lactase,        Laronidase, Lepirudin/hirudin, Mecasermin, Mecasermin rinfabate,        Methoxy Ofatumumab, Natalizumab, Octreotide, Oprelvekin,        Pancreatic amylase, Pancreatic lipase, Papain, Peg-asparaginase,        Peg-doxorubicin HCl, PEG-epoetin-β, Pegfilgrastim,        Peg-Interferon-α2a, Peg-Interferon-α2b, Pegloticase,        Pegvisomant, Phenylalanine ammonia-lyase (PAL), Protein C,        Rasburicase (uricase), Sacrosidase, Salmon calcitonin,        Sargramostim, Streptokinase, Tenecteplase, Teriparatide,        Tocilizumab (atlizumab), Trastuzumab, Type 1 alpha-interferon,        Ustekinumab, and vW factor; provided that interferon (interferon        alpha 2, interferon alpha 5, interferon alpha 6, or consensus        interferon), Ribavirin, Nexavar/Sorafenib, Erbitus/Cetuximab,        Avastatin/bevacizumab, and Herceptin/trastuzumab are excluded        from the scope of Formula 2 when m′+m″ equals 1 and Z is DOM        26h-196-61 or another of the liver-targeting molecules described        in US 2013/0078216.        -   Especially where X is Abciximab, Adalimumab, Agalsidase            alfa, Agalsidase beta, Aldeslukin, Alglucosidase alfa,            Factor VIII, Factor IX, Infliximab, L-asparaginase,            Laronidase, Natalizumab, Octreotide, Phenylalanine            ammonia-lyase (PAL), or Rasburicase (uricase).            -   Particularly where X is Factor VIII, Factor IX, uricase,                PAL or asparaginase.    -   X is a self-antigen polypeptide selected for treating type 1        diabetes mellitus, pediatric multiple sclerosis, juvenile        rheumatoid arthritis, celiac disease, or alopecia universalis.        -   Especially where X is a self-antigen polypeptide selected            for treating new onset type 1 diabetes mellitus, pediatric            multiple sclerosis or celiac disease.    -   X is a foreign antigen to which patients develop an unwanted        immune response        -   From peanut, including conarachin (Ara h 1).        -   From wheat, including Alpha-gliadin “33-mer” native (SEQ ID            NO:24), Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25),            Alpha-gliadin (SEQ ID NO:26) and Omega-gliadin (SEQ ID            NO:27).        -   From cat, including Fel d 1A (UNIPROT P30438) and Cat            albumin (UNIPROT P49064).        -   From dog, including Can f 1 (UNIPROT 018873) and Dog albumin            (UNIPROT P49822).    -   X s is a foreign transplant antigen against which transplant        recipients develop an unwanted immune response, e.g. a human        leukocyte antigen protein.    -   X is an antibody, antibody fragment or ligand that specifically        binds a circulating protein or peptide or antibody, which        circulating protein or peptide or antibody gives rise to        transplant rejection, immune response against a therapeutic        agent, autoimmune disease, and/or allergy.        -   Especially where X binds an endogenous circulating protein            or peptide or antibody.    -   Y is Gly₃Ser.    -   Z is Anti-ASGPR Dom26h-196-61 or a conservative substitution.    -   m′+m″ equals 1 or 2 and X is a full length protein, including        protein therapeutics.    -   m′+m″ equals 1 or 2 where m′ is 1 and m″ is 0 or 1.    -   m′+m″ equals 2 to about 10 where X is a group of self-antigen        peptides (epitopes) known to be associated with a particular        autoimmune disease or for treatment of genetically diverse        target populations.        -   m′+m″ equals about 4 to 7 where the autoimmune disease is            multiple sclerosis and X is indpendently selected from:            MBP13-32 (SEQ ID NO:11), MBP83-99 (SEQ ID NO:12), MBP111-129            (SEQ ID NO:13), MBP146-170 (SEQ ID NO:14), MOG1-20 (SEQ ID            NO:15), MOG35-55 (SEQ ID NO:16) and PLP139-154 (SEQ ID            NO:17).            -   m′+m″ equals 7 and X is, respectively, MBP13-32 (SEQ ID                NO:11), MBP83-99 (SEQ ID NO:12), MBP111-129 (SEQ ID                NO:13), MBP146-170 (SEQ ID NO:14), MOG1-20 (SEQ ID                NO:15), MOG35-55 (SEQ ID NO:16) and PLP139-154 (SEQ ID                NO:17).

As with the above discussion regarding Formula 1, each of theabove-described groups and sub-groups for Formula 2 are individuallypreferred and can be combined to describe further preferred aspects ofthe disclosure.

Utility, Testing and Administration

General Utility

The compositions of the disclosure find use in a variety of applicationsincluding, as will be appreciated by those in the art, treatment oftransplant rejection, immune response against a therapeutic agent,autoimmune disease, and food allergy.

In a preferred embodiment, the compositions of the disclosure are usedto modulate, particularly down-regulate, antigen-specific undesirableimmune response.

The compositions of the disclosure are useful to bind and clear from thecirculation specific undesired proteins, including antibodiesendogenously generated in a patient (i.e., not exogenous antibodiesadministered to a patient), peptides and the like, which causeautoimmunity and associated pathologies, allergy, inflammatory immuneresponses, and anaphylaxis.

In the present disclosure, antigens are targeted to the liver forpresentation via antigen-presenting cells to specifically down-regulatethe immune system or for clearance of unwanted circulating proteins.This is distinct from previous uses of liver targeting, for example asdescribed in US 2013/0078216, where the purpose of liver-targetingmolecules such as DOM26h-196-61 was the delivery of therapeutic agentsto treat liver diseases such as fibrosis, hepatitis, Cirrhosis and livercancer.

The present disclosure provides compositions and methods to treatunwanted immune response to self-antigens and foreign antigens,including but not limited to: a foreign transplant antigen against whichtransplant recipients develop an unwanted immune response (e.g.,transplant rejection), a foreign antigen to which patients develop anunwanted immune (e.g., allergic or hypersensitivity) response, atherapeutic agent to which patients develop an unwanted immune response(e.g., hypersensitivity and/or reduced therapeutic activity), a selfantigen to which patients develop an unwanted immune response (e.g.,autoimmune disease)

Autoimmune disease states that can be treated using the methods andcompositions provided herein include, but are not limited to: AcuteDisseminated Encephalomyelitis (ADEM); Acute interstital allergicnephritis (drug allergies); Acute necrotizing hemorrhagicleukoencephalitis; Addison's Disease; Alopecia areata; Alopeciauniversalis; Ankylosing Spondylitis; Arthritis, juvenile; Arthritis,psoriatic; Arthritis, rheumatoid; Atopic Dermatitis; Autoimmune aplasticanemia; Autoimmune gastritis; Autoimmune hepatitis; Autoimmunehypophysitis; Autoimmune oophoritis; Autoimmune orchitis; Autoimmunepolyendocrine syndrome type 1; Autoimmune polyendocrine syndrome type 2;Autoimmune thyroiditis; Behcet's disease; Bronchiolitis obliterans;Bullous pemphigoid; Celiac disease; Churg-Strauss syndrome; Chronicinflammatory demyelinating polyneuropathy; Cicatricial pemphigoid;Crohn's disease; Coxsackie myocarditis; Dermatitis herpetiformisDuhring; Diabetes mellitus (Type 1); Erythema nodosum; Epidermolysisbullosa acquisita, Giant cell arteritis (temporal arteritis); Giant cellmyocarditis; Goodpasture's syndrome; Graves' disease; Guillain-Barresyndrome; Hashimoto's encephalitis; Hashimoto's thyroiditis;IgG4-related sclerosing disease; Lambert-Eaton syndrome; Mixedconnective tissue disease; Mucha-Habermann disease; Multiple sclerosis;Myasthenia gravis; Optic neuritis; Neuromyelitis optica; Pemphigusvulgaris and variants; Pernicious angemis; Pituitary autoimmune disease;Polymyositis; Postpericardiotomy syndrome; Premature ovarian failure;Primary Biliary Cirrhosis; Primary sclerosing cholangitis; Psoriasis;Rheumatic heart disease; Sjogren's syndrome; Systemic lupuserythematosus; Systemic sclerosis; Ulcerative colitis; Undifferentiatedconnective tissue disease (UCTD); Uveitis; Vitiligo; and Wegener'sgranulomatosis.

A particular group of autoimmune disease states that can be treatedusing the methods and compositions provided herein include, but are notlimited to: Acute necrotizing hemorrhagic leukoencephalitis; Addison'sDisease; Arthritis, psoriatic; Arthritis, rheumatoid; Autoimmuneaplastic anemia; Autoimmune hypophysitis; Autoimmune gastritis;Autoimmune polyendocrine syndrome type 1; Bullous pemphigoid; Celiacdisease; Coxsackie myocarditis; Dermatitis herpetiformis Duhring;Diabetes mellitus (Type 1); Epidermolysis bullosa acquisita; Giant cellmyocarditis; Goodpasture's syndrome; Graves' disease; Hashimoto'sthyroiditis; Mixed connective tissue disease; Multiple sclerosis;Myasthenia gravis; Neuromyelitis optica; Pernicious angemis; Pemphigusvulgaris and variants; Pituitary autoimmune disease; Premature ovarianfailure; Rheumatic heart disease; Systemic sclerosis; Sjogren'ssyndrome; Systemic lupus erythematosus; and Vitiligo.

In the embodiments employing an antigen against which an unwanted immuneresponse is developed, such as food antigens, treatment can be providedfor reactions against, for example: peanut, apple, milk, egg whites, eggyolks, mustard, celery, shrimp, wheat (and other cereals), strawberryand banana.

As will be appreciated by those skilled in the art, a patient can betested to identify a foreign antigen against which an unwanted immuneresponse has developed, and a composition of the disclosure can bedeveloped based on that antigen.

Testing

In establishing the utiity of the compositions and methods of thedisclosure, specificity in binding to antigen-presenting cells in theliver (particularly binding to hepatocytes and specifically ASGPR)should initially be determined. This can be accomplished, for example,by employing a marker (such as the fluorescent marker phycoerythrin(“PE”)) in a composition of the disclosure. The composition isadministered to suitable experimental subjects. Controls, e.g.,unconjugated PE or vehicle (saline) are administered to other group(s)of subjects. The composition and controls are allowed to circulate for aperiod of 1 to 5 hours, after which the spleens and livers of thesubjects are harvested and measured for fluorescence. The specific cellsin which fluorescence is found can be subsequently identified.Compositions of the disclosure, when tested in this manner, show higherlevels of concentration in the antigen-presenting cells of the liver ascompared with unconjugated PE or vehicle.

Effectiveness in immune modulation can be tested by measuring theproliferation of OT-1 CD8⁺ cells (transplanted into host mice) inresponse to the administration of a composition of the disclosureincorporating a known antigen, such as ovalbumin (“OVA”), as comparedwith administration of the antigen alone or just vehicle. Compositionsof the disclosure, when tested in this manner, show an increase of OT1cell proliferation as compared with antigen alone or vehicle,demonstrating increased CD8+ T-cell cross-priming. To distinguish Tcells being expanded into a functional effector phenotype from thosebeing expanded and deleted, the proliferating OT-1 CD8⁺ T cells can bephenotypically analyzed for molecular signitures of exhaustion [such asprogrammed death-1 (PD-1), FasL, and others], as well as annexin-V as ahallmark of apoptosis and thus deletion. The OT-1 CD8⁺ T cells can alsobe assessed for their responsiveness to an antigen challenge withadjuvant in order to demonstrate functional non-responsiveness, and thusimmune tolerance, towards the antigen. To do so, the cells are analyzedfor inflammatory signatures after administration of compositions of thedisclosure into host mice followed by an antigen challenge. Compositionsof the disclosure when tested in this manner demonstrate very low (e.g.,background) levels of inflammatory OT-1 CD8⁺ T cell responses towardsOVA, thus demonstrating immune tolerance.

Humoral immune response can be tested by administering a composition ofthe disclosure incorporating a known antigen, such as OVA, as comparedwith the administration of the antigen alone or just vehicle, andmeasuring the levels of resulting antibodies. Compositions of thedisclosure when tested in this manner show very low (e.g., background)levels of antibody formation responsive to their administration and theadministration of vehicle, with significantly higher levels of antibodyformation responsive to administration of the antigen.

Effectiveness in tolerization against an antigen can be tested as abovewith reference to humoral immune response, where several weeks followingtreatment(s) with a composition of the disclosure a group of subjects ischallenged by administration of the antigen alone, followed by measuringthe levels of antibodies to the antigen. Compositions of the disclosurewhen tested in this manner show low levels of antibody formationresponsive to challenge with the antigen in groups pretreated with suchcompositions as compared to groups that are not pretreated.

Disease-focused experimental models are well known to those skilled inthe art and include the NOD (or non-obese diabetic) mouse model ofautoimmunity and tolerance and the EAE (experimental autoimmuneencephalomyelitis) model for the human inflammatory demyelinatingdisease, multiple sclerosis. In particular, the NOD mouse developsspontaneous autoimmune diabetes (similar to type 1a diabetes in humans).Groups of NOD mice are treated with test compound or a negative control,followed by measurement of BLOOD GLUCOSE. Successful treatmentcorresponds to likelihood of treating diabetes in humans or proof ofmechanism for approaches to the treatment of other autoimmune diseases.(See, e.g., Anderson and Bluestone, Annu. Rev. Immunol. 2005;23:447-85.)

Administration

The compositions of the disclosure are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. Administration of the compounds ofthe disclosure or the pharmaceutically acceptable salts thereof can bevia any of the accepted modes of administration for agents that servesimilar utilities.

While human dosage levels have yet to be optimized for the compounds ofthe disclosure, these can initially be extrapolated from the about 10 μgto 100 μg doses administered for mice. Generally, an individual humandose is from about 0.01 to 2.0 mg/kg of body weight, preferably about0.1 to 1.5 mg/kg of body weight, and most preferably about 0.3 to 1.0mg/kg of body weight. Treatment can be administered for a single day ora period of days, and can be repeated at intervals of several days, oneor several weeks, or one or several months. Administration can be as asingle dose (e.g., as a bolus) or as an initial bolus followed bycontinuous infusion of the remaining portion of a complete dose overtime, e.g., 1 to 7 days. The amount of active compound administeredwill, of course, be dependent on any or all of the following: thesubject and disease state being treated, the severity of the affliction,the manner and schedule of administration and the judgment of theprescribing physician. It will also be appreciated that amountsadministered will depend upon the molecular weight of the antigen,antibody, antibody fragment or ligand as well as the size of the linker.

The compositions of the disclosure can be administered either alone orin combination with other pharmaceutically acceptable excipients. Whileall typical routes of administration are contemplated, it is presentlypreferred to provide liquid dosage forms suitable for injection. Theformulations will typically include a conventional pharmaceuticalcarrier or excipient and a composition of the disclosure or apharmaceutically acceptable salt thereof. In addition, thesecompositions can include other medicinal agents, pharmaceutical agents,carriers, and the like, including, but not limited to the therapeuticprotein, peptide, antibody or antibody-like molecule corresponding tothe antigen (X) employed in the composition of the disclosure, and otheractive agents that can act as immune-modulating agents and morespecifically can have inhibitory effects on B-cells, includinganti-folates, immune suppressants, cyostatics, mitotic inhibitors, andanti-metabolites, or combinations thereof.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 95%,preferably about 0.5% to 50%, by weight of a composition of thedisclosure, the remainder being suitable pharmaceutical excipients,carriers, etc. Dosage forms or compositions containing active ingredientin the range of 0.005% to 95% with the balance made up from non-toxiccarrier can be prepared.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active composition of thedisclosure (e.g., a lyophilized powder) and optional pharmaceuticaladjuvants in a carrier, such as, for example, water (water forinjection), saline, aqueous dextrose, glycerol, glycols, ethanol or thelike (excluding galactoses), to thereby form a solution or suspension.If desired, the pharmaceutical composition to be administered can alsocontain minor amounts of nontoxic auxiliary substances such as wettingagents, emulsifying agents, stabilizing agents, solubilizing agents, pHbuffering agents and the like, for example, sodium acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine acetate and triethanolamine oleate, etc., osmolytes,amino acids, sugars and carbohydrates, proteins and polymers, salts,surfactants, chelators and antioxidants, preservatives, and specificligands. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington: The Science and Practice of Pharmacy, Pharmaceutical Press,22nd Edition, 2012. The composition or formulation to be administeredwill, in any event, contain a quantity of the active compound in anamount effective to treat the symptoms of the subject being treated.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described disclosure, as well as to set forth the best modescontemplated for carrying out various aspects of the disclosure. It isunderstood that these examples in no way serve to limit the true scopeof this disclosure, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1 F1aA-OVA-m₄-n₈₀ (or F1a-OVA-m₄-n₈₀-2NGAL)

1A. Formula 103′ where X′ is OVA and m is 4

In an endotoxin-free tube, OVA (5.0 mg, 0.00012 mmol) was added to 100μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg ofTraut's Reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl(0.00119 mmol) of the Traut's Reagent solution so obtained was added tothe stirred solution of OVA with continued stirring. After 1 hour,excess Traut's Reagent was removed using a centrifugal size exclusioncolumn to afford the corresponding product of Formula 103′.

1B. Formula 106A where n is 80

In an endotoxin-free tube, galactosamine (10.0 mg, 0.04638 mmol) wasdissolved with stirring in 100 μl of pH 8.0 PBS containing 5 mM EDTA.Pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104 where n is80) (16.23 mg, 0.00464 mmol) dissolved in 100 μl of pH 7.0 PBS was addedto the stirring solution of galactosamine. After 1 hour, the resultingpyridyl dithiol-poly(ethylene glycol)-N-acetylgalactosamine (Formula106A) was ready to be used without further purification.

1C. Formula 1 aA where X′ is OVA, m is 4, n is 80 (and Z′ is C2galactosamine)

The purified OVA-Traut conjugate of Formula 103′ prepared in Example 1Awas added directly to the stirring product of Formula 106A prepared inExample 1B. After 1 hour, the resulting product of Formula 1a waspurified by passing the reaction mixture through a centrifugal sizeexclusion column. Characterization (UHPLC SEC, gel electrophoresis)confirmed product identity. (See FIG. 5 .)

1 D. Other Compounds of Formula 103′

By following the procedure described in Example 1A and substituting OVAwith the following:

-   -   Abciximab,    -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   Insulin (SEQ ID NO:5),    -   GAD-65 (SEQ ID NO:6),    -   IGRP (SEQ ID NO:7)    -   MBP (SEQ ID NO:8),    -   MOG (SEQ ID NO:9),    -   PLP (SEQ ID NO:10),    -   MBP13-32 (SEQ ID NO:11),    -   MBP83-99 (SEQ ID NO:12),    -   MBP111-129 (SEQ ID NO:13),    -   MBP146-170 (SEQ ID NO:14),    -   MOG1-20 (SEQ ID NO:15),    -   MOG35-55 (SEQ ID NO:16),    -   PLP139-154 (SEQ ID NO:17),    -   MART1 (SEQ ID NO:18),    -   Tyrosinase (SEQ ID NO:19),    -   PMEL (SEQ ID NO:20),    -   Aquaporin-4 (SEQ ID NO:21),    -   S-arrestin (SEQ ID NO:22),    -   IRBP (SEQ ID NO:23),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:24),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25),    -   Alpha-gliadin (SEQ ID NO:26),    -   Omega-gliadin (SEQ ID NO:27),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE example (UNIPROT P18577),        there are obtained the following corresponding compounds of        Formula 103′ where:    -   X is Abciximab and m is 10,    -   X is Adalimumab and m is 11,    -   X is Agalsidase alfa and m is 14,    -   X is Agalsidase beta and m is 14,    -   X is Aldeslukin and m is 6,    -   X is Alglucosidase alfa and m is 13,    -   X is Factor VIII and m is 100,    -   X is Factor IX and m is 18,    -   X is L-asparaginase and m is 5,    -   X is Laronidase and m is 7,    -   X is Octreotide and m is 1,    -   X is Phenylalanine ammonia-lyase and m is 12,    -   X is Rasburicase and m is 12,    -   X is Insulin (SEQ ID NO:5) and m is 2,    -   X is GAD-65 (SEQ ID NO:6) and m is 8,    -   X is IGRP (SEQ ID NO:7) and m is 7,    -   X is MBP (SEQ ID NO:8) and m is 6,    -   X is MOG (SEQ ID NO:9) and m is 5,    -   X is PLP (SEQ ID NO:10) and m is 8,    -   X is MBP13-32 (SEQ ID NO:11) and m is 1,    -   X is MBP83-99 (SEQ ID NO:12) and m is 1,    -   X is MBP111-129 (SEQ ID NO:13) and m is 1,    -   X is MBP146-170 (SEQ ID NO:14) and m is 2,    -   X is MOG1-20 (SEQ ID NO:15) and m is 1,    -   X is MOG35-55 (SEQ ID NO:16) and m is 2,    -   X is PLP139-154 (SEQ ID NO:17) and m is 3,    -   X is MART1 (SEQ ID NO:18) and m is 4,    -   X is Tyrosinase (SEQ ID NO:19) and m is 8,    -   X is PMEL (SEQ ID NO:20) and m is 5,    -   X is Aquaporin-4 (SEQ ID NO:21) and m is 4,    -   X is S-arrestin (SEQ ID NO:22) and m is 12,    -   X is IRBP (SEQ ID NO:23) and m is 21,    -   X is Conarachin and m is 21,    -   X is Alpha-gliadin “33-mer” native (SEQ ID NO:24) and m is 1,    -   X is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25) and m is        1,    -   X is Alpha-gliadin (SEQ ID NO:26) and m is 1,    -   X is Omega-gliadin (SEQ ID NO:27) and m is 1,    -   X is Fel d 1 and m is 4,    -   X is Cat albumin and m is 16,    -   X is Can f 1 and m is 6,    -   X is Dog albumin and m is 23, and    -   X is RhCE example and m is 10.

1E. Other Compounds of Formula 1 aA

By following the procedure described in Example 1C and substituting thecompounds of Formula 103′, for example as obtained in Example 1D, thereare obtained the following corresponding compounds of Formula 1 aA:

-   -   F1aA-Abciximab-m₁₀-n₈₀,    -   F1aA-Adalimumab-m₁₁-n₈₀,    -   F1aA-Agalsidase alfa-m₁₄-n₈₀,    -   F1aA-Agalsidase beta-m₁₄-n₈₀,    -   F1aA-Aldeslukin-m₈-n₈₀,    -   F1aA-Alglucosidase alfa-m₁₃-n₈₀,    -   F1aA-Factor VIII-m₁₀₀-n₈₀,    -   F1aA-Factor IX-m₁₈-n₈₀,    -   F1aA-L-asparaginase-m₅-n₈₀,    -   F1aA-Laronidase-m₇-n₈₀,    -   F1aA-Octreotide-m₁-n₈₀,    -   F1aA-Phenylalanine ammonia-lyase-m₁₂-n₈₀,    -   F1aA-Rasburicase-m₁₂-n₈₀,    -   F1aA-Insulin-m₂-n₈₀,    -   F1aA-GAD-65-m₈-n₈₀,    -   F1aA-IGRP-m₇-n₈₀,    -   F1aA-MBP-m₈-n₈₀,    -   F1aA-MOG-m₅-n₈₀,    -   F1aA-PLP-m₈-n₈₀,    -   F1aA-MBP13-32-m₁-n₈₀,    -   F1aA-MBP83-99-m₁-n₈₀,    -   F1aA-MBP111-129-m₁-n₈₀,    -   F1aA-MBP146-170-m₂-n₈₀,    -   F1aA-MOG1-20-m₁-n₈₀,    -   F1aA-MOG35-55-m₂-n₈₀,    -   F1aA-PLP139-154-m₃-n₈₀,    -   F1aA-MART1-m₄-n₈₀,    -   F1aA-Tyrosinase-m₈-n₈₀,    -   F1aA-PMEL-m₅-n₈₀,    -   F1aA-Aquaporin-4-m₄-n₈₀,    -   F1aA-S-arrestin-m₁₂-n₈₀,    -   F1aA-IRBP-m₂₁-n₈₀,    -   F1aA-Conarachin-m₂₁-n₈₀,    -   F1aA-Alpha-gliadin “33-mer” native-m₁-n₈₀,    -   F1aA-Alpha-gliadin “33-mer” deamidated-m₁-n₈₀,    -   F1aA-Alpha-gliadin-m₁-n₈₀,    -   F1aA-Omega-gliadin-m₁-n₈₀,    -   F1aA-Fel d 1-m₄-n₈₀,    -   F1aA-Cat albumin-m₁₆-n₈₀,    -   F1aA-Can f 1-m₆-n₈₀,    -   F1aA-Dog albumin-m₂₃-n₈₀, and    -   F1aA-RhCE-m₁₀-n₈₀.

1F. Other Compounds of Formula 106A

By following the procedure described in Example 1B and substituting thepyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104 where n is80) with the following:

-   -   Formula 104 where n is 12,    -   Formula 104 where n is 33,    -   Formula 104 where n is 40,    -   Formula 104 where n is 43,    -   Formula 104 where n is 50,    -   Formula 104 where n is 60,    -   Formula 104 where n is 75, and    -   Formula 104 where n is 80,        there are obtained the following corresponding compounds of        Formula 106A where:    -   n is 12,    -   n is 33,    -   n is 40,    -   n is 43,    -   n is 50,    -   n is 60,    -   n is 75, and    -   n is 84,

1G. Other Compounds of Formula 1aA

By following the procedure described in Example 1E and substituting thecompound of Formula 106A with the compounds obtained in Example 1F,there are obtained the corresponding compounds of Formula 1 aA where nis 12, 33, 40, 43, 50, 60, 75 and 84, such as:

-   -   F1aA-Insulin-m₂-n₁₂,    -   F1aA-Insulin-m₂-n₃₃,    -   F1aA-Insulin-m₂-n₄₀,    -   F1aA-Insulin-m₂-n₄₃,    -   F1aA-Insulin-m₂-n₅₀,    -   F1aA-Insulin-m₂-n₆₀,    -   F1aA-Insulin-m₂-n₇₅, and    -   F1aA-Insulin-m₂-n₈₄.

Example 2 F1b-OVA-m-n₄-p₃₄-2NAcGAL

2A. Formula 103′ where X′ is Ovalbumin and m is 1

In an endotoxin-free tube, OVA (6.5 mg, 0.000155 mmol) was added to 200μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg ofTaut's Reagent was dissolved in 100 μl of pH 7.0 PBS, and 43 μl (0.00310mmol) of the Traut's Reagent solution so obtained was added to thestirred solution of OVA with continued stirring. After 1 hour,non-reacted Traut's Reagent was removed using a centrifugal sizeexclusion column to afford the product of Formula 103′.

2B. Formula 1b where X′ is Ovalbumin, m is 1, n is 4, p is 34, R⁹ is adirect bond and Z″ is 2NAcGAL

In a micro centrifuge tube, poly(Galactosamine Methacrylate)-(pyridyldisulfide) (Formula 201) (20.0 mg, 0.0020 mmol) was solubilized in 50 μlof pH 8.0 PBS containing 5 mM EDTA. To this was added the purifiedOVA-Traut product from Example 2A followed by stirring for 1 hour. Theresulting product of Formula 1b was purified by passing the reactionmixture through a centrifugal size exclusion column. Characterization(UHPLC SEC, gel electrophoresis) confirmed the identity of the product.(See FIG. 5 .)

2C. Other Compounds of Formula 1b

By following the procedure described in Example 2B and substituting thecompounds of Formula 103′, for example as obtained in Example 1 D, thereare obtained the following corresponding compounds of Formula 1 b:

-   -   F1b-Abciximab-m₁₀-n₄-p₃₄-2NAcGAL,    -   F1b-Adalimumab-m₁₁-n₄-p₃₄-2NAcGAL,    -   F1b-Agalsidase alfa-m₁₄-n₄-p₃₄-2NAcGAL,    -   F1b-Agalsidase beta-m₁₄-n₄-p₃₄-2NAcGAL,    -   F1b-Aldeslukin-m₈-n₄-p₃₄-2NAcGAL,    -   F1b-Alglucosidase alfa-m₁₃-n₄-p₃₄-2NAcGAL,    -   F1b-Factor VIII-m₁₀₀-n₄-p₃₄-2NAcGAL,    -   F1b-Factor IX-m₁₈-n₄-p₃₄-2NAcGAL,    -   F1b-L-asparaginase-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-Laronidase-m₇-n₄-p₃₄-2NAcGAL,    -   F1b-Octreotide-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Phenylalanine ammonia-lyase-m₁₂-n₄-p₃₄-2NAcGAL,    -   F1b-Rasburicase-m₁₂-n₄-p₃₄-2NAcGAL,    -   F1b-Insulin-m₂-n₄-p₃₄-2NAcGAL,    -   F1b-GAD-65-m₈-n₄-p₃₄-2NAcGAL,    -   F1b-IGRP-m₇-n₄-p₃₄-2NAcGAL,    -   F1b-MBP-m₆-n₄-p₃₄-2NAcGAL,    -   F1b-MOG-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-PLP-m₈-n₄-p₃₄-2NAcGAL,    -   F1b-MBP13-32-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-MBP83-99-m₁-n₄-p₃₄-2NACGAL,    -   F1b-MBP111-129-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-MBP146-170-m₂-n₄-p₃₄-2NAcGAL,    -   F1b-MOG1-20-m₁-n₄-p₃₄-2NACGAL,    -   F1b-MOG35-55-m₂-n₄-p₃₄-2NAcGAL,    -   F1b-PLP139-154-m₃-n₄-p₃₄-2NAcGAL,    -   F1b-MART1-m₄-n₄-p₃₄-2NAcGAL,    -   F1b-Tyrosinase-m₈-n₄-p₃₄-2NAcGAL,    -   F1b-PMEL-m₅-n₄-p₃₄-2NAcGAL,    -   F1b-Aquaporin-4-m₄-n₄-p₃₄-2NAcGAL,    -   F1b-S-arrestin-m₁₂-n₄-p₃₄-2NAcGAL,    -   F1b-IRBP-m₂₁-n₄-p₃₄-2NAcGAL,    -   F1b-Conarachin-m₂₁-n₄-p₃₄-2NACGAL,    -   F1b-Alpha-gliadin “33-mer” native-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Alpha-gliadin “33-mer” deamidated-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Alpha-gliadin-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Omega-gliadin-m₁-n₄-p₃₄-2NAcGAL,    -   F1b-Fel d 1-m₄-n₄-p₃₄-2NAcGAL,    -   F1b-Cat albumin-m₁₆-n₄-p₃₄-2NAcGAL,    -   F1b-Can f 1-m₆-n₄-p₃₄-2NAcGAL,    -   F1b-Dog albumin-m₂₃-n₄-p₃₄-2NAcGAL, and    -   F1b-RhCE-m₁₀-n₄-p₃₄-2NAcGAL.

Example 3 F1f-OVA-m-n₄-p₃₃-2NAcGAL

3A. Formula 1f where X′ is Ovalbumin and m is 1, n is 4, p is 33, R⁹ isa direct bond and Z″ is 2NAcGAL

In an endotoxin-free tube, OVA (4.0 mg, 0.0000952381 mmol) was added to0.1 ml of pH 7.4 PBS and stirred. Separately,poly-(n-Acetylgalactosamine)-p-nitrophenyol carbonate of Formula 601where n is 4 and p is 33 (33.0 mg, 0.002380952 mmol) was added to 100 μlof pH 7.5 PBS and vortexed until dissolved. The two solutions werecombined and the mixture was stirred vigorously for 1 hour. The mixturewas then collected and dialyzed for 3 days against pH 7.4 PBS (30 kDamolecular weight cut off) to afford the product of Formula 1f.

Example 4 F1g-PVA-m₁-p₉₀-2NAcGAL

4A. Formula 1q where X′ is Ovalbumin and m is 1, p is 90, R⁹ is a directbond and Z″ is 2NAcGAL

In an endotoxin-free tube, OVA (5.0 mg, 0.000119048 mmol) was added to0.2 ml of pH 7.4 PBS and stirred. To the stirring solution was added 75mg (0.00297619 mmol) of Poly(Galactosamine Methacrylate)-NHS (Formula701) dissolved in 0.4 ml of pH 7.4 PBS. The mixture was allowed to stirfor 2 hours. The mixture was then collected and dialyzed for 3 daysagainst pH 7.4 PBS (30 kDa molecular weight cut off) to afford theproduct of Formula 1g.

Example 5 F1h-OVA-m₂-n₄₅-p₅₅-q₄-2NAcGAL

5A. Formula 802′ where X′ is Ovalbumin, m is 2 and n is 45

In an endotoxin-free tube, OVA (3.0 mg, 0.0000714286 mmol) was added to150 μl of pH 8.0 PBS containing 5 mM EDTA and stirred.Dibenzocyclooctyne-PEG-(p-nitrophenyl carbonate) (Formula 801) (5.265mg, 0.002142857 mmol) dissolved in DMF was added to the OVA solution andstirred for 1 hour. The excess dibenzocyclooctyne-PEG-(p-nitrophenylcarbonate) was removed using a centrifugal size exclusion column toafford the product of Formula 802′.

5B. Formula 1h where X′ is Ovalbumin, m is 2, n is 45, p is 55, q is 4,R⁸ is CH₂, R⁹ is a direct bond and Z″ is 2NAcGAL

Poly(Galactosamine Methacrylate)-N3 (Formula 803 where p is 55, q is 4and Z″ is N-acetylgalactosamine) (33 mg, 0.002142857 mmol) was dissolvedin 100 μl of pH 7.4 PBS and added to the product of Example 5A withstirring. After 1 hour, the resulting product of Formula 1 h waspurified by centrifugal size exclusion chromatography.

Example 6 F1j-OVA-m₁₀-n₄₅-p₅₅-q₄-2NAcGAL

6A. Formula 103′ where X′ is Ovalbumin and m is 10

In an endotoxin-free tube, OVA (5.0 mg, 0.00019 mmol) was added to 150μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg ofTaut's Reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl (0.0019mmol) of the Traut's Reagent solution so obtained was added to thestirred solution of OVA with continued stirring. After 1 hour,non-reacted Traut's Reagent was removed using a centrifugal sizeexclusion column to afford the product of Formula 103′.

6B. Formula 902″ where X′ is Ovalbumin, m is 10 and n is 45

Dibenzocyclooctyne-PEG-(pyridyl disulfide) (Formula 901 where n is 45)(6.0 mg, 0.00238 mmol) was dissolved in DMF and the resulting solutionwas added to the OVA solution obtained in Example 6A and stirred for 1hour. The excess dibenzocyclooctyne-PEG-(pyridyl disulfide) was removedusing centrifugal size exclusion chromatography to afford the product ofFormula 902″.

6C. Formula 1j where X′ is Ovalbumin, m is 10, n is 45, p is 55, q is 4,R⁸ is CH₂, R⁹ is a direct bond and Z″ is 2NAcGAL

Poly(Galactosamine Methacrylate)-N3 (Formula 803 where p is 55, q is 4and Z″ is N-acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in150 μl of pH 7.4 PBS and added to the product of Example 6B withstirring. After 1 hour, the resulting product of Formula 1j was purified(excess p(GMA)-N3 removed) by centrifugal size exclusion chromatography.Characterization (UHPLC SEC, gel electrophoresis) confirmed the identityof the product.

Example 7 F1L-OVA-m₂-n₈₀-p₅₅-q₄-2NAcGAL

7A. Formula 1002 where X′ is Ovalbumin, m is 2 and n is 80

Dibenzocyclooctyne-PEG-(pyridyl disulfide) (Formula 1001 where n is 80)(9.0 mg, 0.00238 mmol) was dissolved in DMF and the resulting solutionwas added to a purified OVA solution of Formula 103′ (where X′ isOvalbumin and m is 2), for example prepared as described in Example 6Aand stirred for 1 hour. The excess dibenzocyclooctyne-PEG-(pyridyldisulfide) was removed using centrifugal size exclusion chromatographyto afford the product of Formula 1002.

7B. Formula 1L where X′ is Ovalbumin, m is 2, n is 80, p is 55, q is 4,R⁸ is CH₂, R⁹ is a direct bond and Z″ is 2NAcGAL

Poly(Galactosamine Methacrylate)-N3 (Formula 803 where p is 55, q is 4and Z″ is N-Acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in150 μl of pH 7.4 PBS and added to the product of Example 7A withstirring. After 1 hour, the resulting product of Formula 1L was purified(excess poly(Galactosamine Methacrylate)-N3 removed) by centrifugal sizeexclusion chromatography. Characterization (UHPLC SEC, gelelectrophoresis) confirmed the identity of the product.

Example 8 Preparation of poly(Galactosamine methacrylate) Polymers

8A. Galactosamine Methacrylate

To stirred galactosamine hydrochloride (2.15 g, 10.0 mmol) was added 0.5M sodium methoxide (22 ml, 11.0 mmol). After 30 minutes, methacrylateanhydride (14.694 g, 11.0 mmol) was added and stirring continued for 4hours. The resulting galactosamine methacrylate was loaded onto silicagel via rotovap and purified via column chromatography using DCM:MeOH(85:15).

8B. Formula 201 where n is 4 and p is 30

Galactose methacrylate (600 mg, 2.43 mmol),2-(2-(2-(2-(pyridin-2-yldisulfanyl)ethoxy)ethoxy)ethoxy)ethyl2-((phenylcarbonothioyl)thio)acetate (44.8 mg, 0.081 mmol) and AIBN(3.174089069 mg, 0.016 mmol) were added to 1.5 ml of DMF in a SchlenkFlask. The reaction mixture was subjected to 4 freeze-thaw chycles andthen stirred at 70° C. for 6 hours. The desired polymer product ofFormula 201 was precipitated in 12 ml of methanol, and excess solventwas removed under reduced pressure.

Example 9 Preparation of F1aA-PE-m₃-n₈₀

9A. Formula 103′ where X′ is Phycoerythrin

In an endotoxin-free tube, phycoerythrin (“PE”) (purchased from Pierce)(200 μl, 0.000004 mmol) was added to 50 μl of pH 8.0 PBS containing 5 mMEDTA and stirred. Separately, 1 mg of Taut's Reagent was dissolved in100 μl of pH 7.0 PBS, and 2 μl (0.00013 mmol) of the Traut's Reagentsolution so obtained was added to the stirred solution of PE withcontinued stirring. After 1 hour, excess Traut's Reagent was removedusing a centrifugal size exclusion column to afford the product ofFormula 103′.

9B. Formula 106A where n is 80

In an endotoxin-free tube, galactosamine (7.0 mg, 0.03246 mmol) wasdissolved with stirring in 100 μl of pH 8.0 PBS containing 5 mM EDTA.Pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104 where n is80) (16.23 mg, 0.00464 mmol) dissolved in 50 μl of pH 7.0 PBS was addedto the stirring solution of galactosamine. After 1 hour, the resultingproduct of Formula 106A was ready to be used without furtherpurification.

9C. Formula 1a where X′ is Phycoerythrin, m is 3, n is 80 and Z′ isgalactosamine

The purified PE-Traut conjugates prepared in Example 9A were addeddirectly to the stirring product of Formula 106A prepared in Example 9B.After 1 hour, the resulting product of Formula 1 a was purified bypassing the reaction mixture through a centrifugal size exclusioncolumn. Characterization (UHPLC SEC, gel electrophoresis) confirmed theidentity of the product.

Example 10 OVA-DOM

10A. Preparation of Expression Vector

The mammalian cell expression vector pSecTag A was purchased from LifeTechnologies. The gene encoding the anti-ASGPR domain antibody,Dom26h-196-61, herein referred to as “DOM”, was purchased as acodon-optimized sequence for protein expression in human cells, from theprovider Genscript. Sequences encoding OVA, DOM, the flexible linkerGly₃Ser, and the 6×His tag were cloned into mammalian expression vectorpSecTag A, C-terminal to the Ig κ-chain secretion leader sequence, bysite-directed mutagenesis, following a variation of the QuikChangeprotocol (Geiser, et al.)

10B. Expression and Purification of Formula 2 where m′ is 1, m″ is 0, Xis Ovalbumin, Y is Gly₃Ser, Z is Anti-ASGPR Dom26h-196-61

HEK293 cells were transiently transfected with modified pSecTag Avectors, prepared for example as described in Example 10A, usingpolyethylenimine. Transfected cells were cultured in FreeStyle 293medium (Life Technologies) supplemented with valproic acid for 7 days,after which the cells were removed by centrifugation and the culturesupernatants were collected and sterilized by filtration. The OVA/DOMfusion proteins of Formula 2 were purified from the culture supernatantsby immobilized metal ion affinity chromatography using a HisTrap Ni²⁺sepharose column (GE Healthcare), followed by size exclusionchromatography using a Superdex 75 column (GE Healthcare), having thefollowing generalized structure:

N-OVA-Gly₃Ser-DOM-Gly₃Ser-6×His-C

and the following amino acid sequence:

(SEQ ID NO: 28)GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSHHHHHH

Protein purity was verified by Coomassie Brilliant Blue staining ofSDS-PAGE gels and anti-6×His tag western blotting. Protein concentrationwas determined using the Beer-Lambert Law, for which the absorbance at280 nm was measured using a NanoDrop 2000 (Thermo Scientific), and themolecular weight (57.3 kDa) and extinction coefficient (57,090 M⁻¹ cm⁻¹)were estimated from the protein's amino acid sequence, using the ExPASyProtParam tool. Endotoxin levels were measured using the HEK-BLUE TLRreporter cell line (Invivogen) according to manufacturer's instructions.

10C. Other Compositions of Formula 2

By following the procedures described in Examples 10A and 10B andsubstituting the pSecTag A vectors accordingly, there were obtained thefollowing fusion proteins of Formula 2:N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C having the following amino acidsequence:

(SEQ ID NO: 29)EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH;and

N-OVA-Gly₃Ser-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C

having the following amino acid sequence:

(SEQ ID NO: 30)GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH.

10D. Other Compositions of Formula 2

By following the procedures described in Examples 10A and 10B andsubstituting for Gly₃Ser the vectors for a linker having animmunoproteosome cleavage site (“IPC”), and for OVA the vectors for:

-   -   MBP13-32 (SEQ ID NO:11),    -   MBP83-99 (SEQ ID NO:12),    -   MBP111-129 (SEQ ID NO:13),    -   MBP146-170 (SEQ ID NO:14),    -   MOG1-20 (SEQ ID NO:15),    -   MOG35-55 (SEQ ID NO:16), and    -   PLP139-154 (SEQ ID NO:17),        or the vectors for:    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25)    -   Alpha-gliadin (SEQ ID NO:26), and    -   Omega-gliadin (SEQ ID NO:27),        there are obtained the following fusion proteins of Formula 2:    -   MBP13-32-IPC-MBP83-99-IPC-MBP111-129-IPC-MBP146-170-IPC-MOG1-20-IPC-MOG35-55-IPC-PLP139-154-IPC-DOM,        and    -   Alpha-gliadin “33-mer”        deamidated-IPC-Alpha-gliadin-IPC-Omega-gliadin-IPC-DOM.

Example 11 Desialylated OVA

11A. Preparation of Expression Vector

The mammalian cell expression vector pSecTag A was purchased from LifeTechnologies. Sequences encoding OVA, the flexible linker Gly₃Ser, andthe 6×His tag were cloned into pSecTag A, C-terminal to the Ig κ-chainsecretion leader sequence, by site-directed mutagenesis, following avariation of the QuikChange protocol (Geiser, et al.)

11B. Expression and Purification of OVA

HEK293 cells were transiently transfected with modified pSecTag Avector, prepared for example as described in Example 10A, usingpolyethylenimine. Transfected cells were cultured in FreeStyle 293medium (Life Technologies) supplemented with valproic acid for 7 days,after which the cells were removed by centrifugation and the culturesupernatants are collected and sterilized by filtration. OVA protein waspurified from the culture supernatant by immobilized metal ion affinitychromatography using a HisTrap Ni2+ sepharose column (GE Healthcare),followed by size exclusion chromatography using a Superdex 75 column (GEHealthcare), having the following structure:

N′-OVA Gly₃Ser 6×His-C′

and the following amino acid sequence:

(SEQ ID NO: 31)GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH

Protein purity was verified by Coomassie Brilliant Blue staining ofSDS-PAGE gels and anti-6×His tag western blotting. Protein concentrationwas determined using the Beer-Lambert Law, for which the absorbance at280 nm was measured using a NanoDrop 2000 (Thermo Scientific), and themolecular weight (43.8 kDa) and extinction coefficient (31,525 M-1 cm-1)were estimated from the protein's amino acid sequence, using the ExPASyProtParam tool.

11C. Desialylation

OVA is desialylated by incubation with neuraminidase for 1 hour at 37°C. (New England Biolabs). Desialylated OVA is purified from the reactionmixture by immobilized metal ion affinity chromatography (e.g., using aHisTrap Ni2+ sepharose column, GE Healthcare), followed by sizeexclusion chromatography (e.g., using a Superdex 75 column, GEHealthcare).

Protein purity is verified by Coomassie Brilliant Blue staining ofSDS-PAGE gels and anti-6×His tag western blotting. Desialylation isverified by Sambucus nigra lectin-based detection of protein sialic acidcontent in western blots (Vector Biolabs) and by a sialic acid-mediatedfluorescence assay (ProZyme). Desialylated protein concentration isdetermined using the Beer-Lambert Law, as described above for theprotein before desialylation. Endotoxin levels are measured using theHEK-Blue TLR4 reporter cell line (Invivogen) according to manufacturer'sinstructions.

Example 12 Hepatic Distribution

12A. F1aA-PE-m₃-n₈₀ was prepared, for example, as described in Example9. A 30 μg/100 μl solution in sterile saline was prepared for injection.

The F1aA-PE-m₃-n₈₀ solution (30 μg) was administered to one of threegroups of C57 black 6 mice 3 per group) via tail vein injection. The twoother groups of mice received an equivalent volume of phycoerythrin in100 μl of saline or saline vehicle. Three hours after administration,the livers and spleens of these animals were harvested and the level ofcellular fluorescents in these organs was determined by flow cytometryas an indication of cellular PE content.

As shown in FIG. 1A-1D, sinusoidal endothelial cells (LSECs),hepatocytes, kupffer cells (KC), and other antigen-presenting cells(APCs) from the livers of mice treated with F1aA-PE-m₃-n₈₀ exhibited atleast a three-fold increase in fluorescence as compared with animalsthat received PE solution. No detectible difference in fluorescence wasfound in spleen cells harvested from the three groups. These resultsconfirm that F1aA-PE-m₃-n₈₀ has sufficient specificity for binding toantigen-presenting cells in the liver.

12B. By following the procedure described in Example 12A andsubstituting F1aA-PE-m₃-n₈₀ with the compounds F1b-PE-m₃-n₄-p₃₄-2NAcGAL,F1f-PE-m₃-n₄-p₃₃-2NAcGAL, F1g-PE-m₃-p₉₀-2NAcGAL,F1h-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL, F1j-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL,F1L-PE-m₃-n₈₀-p₅₅-q₄-2NAcGAL, F1m-PE-m₃-n₈₀-p₃₀-q₄-CMP-2NHAc,F1m-PE-m₃-n₆₂-p₃₀-q-CMP-2OH, F1n-PE-m₃-n₁-p₃₀-q₄-CMP-2NHAc andF1n-PE-m₃-n₃₃-p₃₀-q-CMP-2OH, prepared, for example, as described withreference to Example 9 by substitution for X in Examples 2B, 3, 4, 5B,6B, 7B, 19G, 19L, 20B and 20F, respectively it is confirmed that thecompounds F1aA-PE-m₃-n₈₀ with the compounds F1b-PE-m₃-n₄-p₃₄-2NAcGAL,F1f-PE-m₃-n₄-p₃₃-2NAcGAL, F1g-PE-m₃-p₉₀-2NAcGAL,F1h-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL, F1j-PE-m₃-n₄₅-p₅₅-q₄-2NAcGAL,F1L-PE-m₃-n₈₀-p₅₅-q₄-2NAcGAL, F1m-PE-m₃-n₈₀-p₃₀-q₄-CMP-2NHAc,F1m-PE-m₃-n₆₂-p₃₀-q-CMP-2OH, F1n-PE-m₃-n₁-p₃₀-q₄-CMP-2NHAc andF1n-PE-m₃-n₃₃-p₃₀-q₈-CMP-2OH have sufficient specificity for binding toantigen-presenting cells in the liver.

Example 13 Proliferation of Antigen-specific OT1 CD8⁺ Tcells

13A. F1aA-OVA-m₄-n₈₀ synthesized, for example, as described in Example1, was prepared as a 10 μg/100 μl saline solution for injection. On day0, 10⁶ OT-1 T cells were fluorecently labeled and adoptively transferredinto 3 groups of CD 45.2 mice (5 per group) via tail vein injection. Thenext day (i.e. Day 1), to each of the 3 groups of mice were administed,respectively, 10 μg of F1aA-OVA-m₄-n₈₀, OVA or saline via tail veininjection. On day 6, the animals were sacrificed and the % of splenicproliferating OT-1 cells was determined via florescence activated cellsorting.

The results from this study (see FIG. 2 ) show that the percentage ofproliferating OTI T cells in mice treated with F1aA-OVA-m₄-n₈₀(“Gal-OVA” in FIG. 2 ) was significantly greater than the percentage ofproliferating OTI cells in the spleens of mice treated with OVA orsaline (“naïve” in FIG. 2 ). The increase in OTI cell-proliferationdemonstrates the increased CD8⁺ T-cell cross-priming in animals treatedwith F1aA-OVA-m₄-n₈₀ versus the other therapies. In concert with theresults from Example 12, these results indicate that the ability ofF1aA-OVA-m₄-n₈₀ to target antigens to the liver increases OVApresentation by antigen presenting cells in the liver to OVA-specificOTI T cells.

13B. To distinguish T cells being expanded into a functional effectorphenotype from those being expanded and deleted, the proliferating OTICD8⁺ T cells were analyzed for annexin-V, as a hallmark of apoptosis andthus deletion, as well as the exhaustion marker programmed death-1(PD-1). As shown in FIG. 3A-3B, F1aA-OVA-m₄-n₈₀ (“Gal-OVA” in FIG.3A-3B) induced much higher numbers of annexin-V+ and PD-1⁺ proliferatingOTI CD8⁺ T cells than soluble OVA.

13C. By following the procedure described in Examples 13A and 13B, andsubstituting F1aA-OVA-m₄-ne with the compounds of Formula 1 obtained,for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it isshown the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G induce muchhigher numbers of annexin-V+ and PD-1⁺ proliferating OTI CD8⁺ T cellsthan soluble OVA.

13D. By following the procedure described in Examples 13A and 13B andsubstituting F1aA-OVA-m₄-ne with the compounds of Formulae 1 and 2obtained, for example, as described in Examples 1E, 1G, 2C, 10D, 19I,19L, 20B, 20D and 20F, and substituting OVA with the antigenscorresponding to X (or X′ or X″), respectively, it is shown that thecompounds from Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20Finduce much higher numbers of annexin-V+ and PD-1⁺ proliferating OTICD8⁺ T cells than soluble antigen X.

Example 14 F1aA-OVA-m₄-n₆ does not Induce an OVA-Specific AntibodyResponse

14A. In order to assess the humoral immune response to F1aA-OVA-m₄-ne wetreated mice with a weekly i.v. injection of either F1aA-OVA-m₄-n₈ orOVA, then measured the levels of OVA-specific antibodies in the blood.On day 0, 7, and 14 of the experiment, mice were administered an i.v.injection of 100 μl of saline contining one of the following: 1.) 6 μgof OVA; 2.) 6 μg of F1aA-OVA-m₄-n₈; 3.) 30 μg of OVA; 4.) 30 μg ofF1aA-OVA-m₄-n₈, or 5.) saline alone. Each group contained 5 mice. On day19, the mice were bled via cheek puncture, and the titer of OVA-specificantibodies in each mouse's blood was determined via ELISA. The resultsfor this study show that although mice treated with 6 and 30 μg of OVAhad increased OVA-specific antibody titers, mice treated with both 6 and30 μg of F1aA-OVA-m₄-n₈ (“Gal-OVA” in FIG. 4 ) had blood titers similarto mice treated with saline (i.e. vehicle treated animals) (FIG. 4 ).For example mice treated with 6 and 30 μg of OVA had an average antibodytiter of 3.5 and 2.5, respectively; whereas, mice treated with 6 and 30μg of OVA had an average antibody titer of 0.75 and 0.25, respectively.

14B. By following the procedure described in Example 14A andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formula 1 obtained,for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it isshown that mice treated with the compounds from Examples 3A, 4A, 5B, 6C,7B and 19G have OVA-specific antibody titers similar to mice treatedwith saline.

140. By following the procedure described in Example 14A andsubstituting F1aA-OVA-m₄-n₈ with the compounds of Formulae 1 and 2obtained, for example, as described in Examples 1E, 1G, 2C, 10D, 19I,19L, 20B, 20D and 20F, and substituting OVA with the antigenscorresponding to X (or X′ or X″), respectively, it is shown that micetreated with the compounds from Examples 1E, 1G, 2C, 10D, 191, 19L, 20B,20D and 20F have antigen X-specific antibody titers similar to micetreated with saline.

Example 15 F1aA-OVA-m₄-n₈ Depletes OVA-Specific Antibodies

15A. We treated mice that had different OVA-antibody blood titers (eachmouse had a titer from 0 to 4.5) with an i.v. injection of 20 μg ofF1aA-OVA-m₄-n₈ solubilized in 100 μl saline. Mice were given i.v.injections of F1aA-OVA-m₄-n₈ on days 0, 5, 7, 12, and 14 (Injections ofF1aA-OVA-m₄-n₈ are labeled as “Gal-OVA” and shown as green arrows on thex-axis of FIG. 5 ). In order to determine the ability ofF1aA-OVA-m₄-n_(B) to deplete serum OVA-specific antibodies, the micewere bled on day −1 to establish an intital antibody titer and thensubsequent bleeds were carried out after each injection ofF1aA-OVA-m₄-n₈ on days 2, 6, 9. 13, and 16. The antibody titer for eachmouse was determined via ELISA. The results from this study show thatF1aA-OVA-m₄-n₈ is able to deplete serum antibody levels in mice. Forexample, one day after the first F1aA-OVA-m₄-n_(B) injection (i.e. day2), mice with postivie OVA-antibody titers experience a 5 to 100-folddecrease in serum antibody levels (FIG. 5 ). Our results show thatalthough over the course of the 19 day experiment, antibody titers didincrease for certain mice, the titer levels never reached the initialantibody titer measured on Day −1 and subsequent doeses ofF1aA-OVA-m₄-n₈ were effective in reducing these transient increases inantibody titers. These results demonstrate that F1aA-OVA-m₄-n_(B) hasthe specificity to bind serum OVA-specific antibodies and the kineticsrequired to deplete OVA-specific serum antibodies.

15B. By following the procedure described in Example 15A andsubstituting F1aA-OVA-m₄-ne with the compounds of Formula 1 obtained,for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it isshown that the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G havethe specificity to bind serum OVA-specific antibodies and the kineticsrequired to deplete OVA-specific serum antibodies.

15C. By following the procedure described in Example 15A andsubstituting F1aA-OVA-m₄-ne with the compounds of Formulae 1 and 2obtained, for example, as described in Examples 1E, 1G, 2C, 10D, 19I,19L, 20B, 20D and 20F, and substituting OVA with the antigenscorresponding to X (or X′ or X″), respectively, it is shown that thecompounds from Examples 1E, 1G, 2C, 10D, 191, 19L, 20B, 20D and 20F havethe specificity to bind serum antigen X-specific antibodies and thekinetics required to deplete antigen X-specific serum antibodies.

Example 16 OT-1 Challenge-to-Tolerance Model

16A. Using an established OTI challenge-to-tolerance model (Liu, Iyoda,et al., 2002), the ability of F1aA-OVA-m₄-ne (mGal-OVA),F1b-OVA-m₁-n₄-p₃₄ (pGal-OVA), and N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C(Dom-OVA) to prevent subsequent immune responses to vaccine-mediatedantigen challenge were demonstrated—even with a challenge involving avery strong bacterially-derived adjuvant (i.e. lipopolysaccharide). Totolerize, 233 nmol of either F1aA-OVA-m₄-n₈, F1b-OVA-m₁-n₄-p₃₄,N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C, or soluble OVA were intravenouslyadministered in 100 μl saline at 1 and 6 days following adoptivetransfer of OTI CD8+(CD45.2+) T cells to CD45.1⁺ mice (n=5 mice pergroup). After 9 additional days to allow potential deletion of thetransferred T cells, the recipient mice were then challenged with OVA(10 μg) adjuvanted with lipopolysaccharide (LPS) (50 ng) by intradermalinjection. Characterization of the draining lymph nodes 4 d afterchallenge allowed a determination as to whether or not deletion actuallytook place.

16B. Intravenous administration of F1aA-OVA-m₄-n₈, F1b-OVA-m₁-n₄-p₃₄,and N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C resulted in profound reductions inOTI CD8⁺ T cell populations in the draining lymph nodes as compared tomice treated with unmodified OVA prior to antigen challenge with LPS,demonstrating deletional tolerance. For example, FIG. 6 shows that thedraining lymph nodes from mice treated with either F1aA-OVA-m₄-ne(mGal-OVA), F1b-OVA-m₁-n₄-p₃₄ (pGal-OVA), andN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C (Dom-OVA) contained over 9-fold fewerOTI CD8⁺ T cells as compared to OVA-treated mice, and more than 43-foldfewer than the challenge control mice that did not receive intravenousinjections of antigen; responses in spleen cells were similar. Theseresults demonstrate that F1aA-OVA-m₄-n₈, F1b-OVA-m₁-n₄-p₃₄, andN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C mitigated an OVA-specific immuneresponse after adjuvented OVA challenge.

160. By following the procedure described in Examples 16A and B, andsubstituting F1aA-OVA-m₄-n₈, F1b-OVA-m₁-n₄-p₃₄, andN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C with the compounds of Formula 1obtained, for example, as described in Examples 3A, 4A, 5B, 6C, 7B and19G, it is shown that the compounds from Examples 3A, 4A, 5B, 6C, 7B and19G mitigate an OVA-specific immune response after adjuvented OVAchallenge.

16D. By following the procedure described in Examples 16A and B, andsubstituting F1aA-OVA-m₄-n₈, F1b-OVA-m₁-n₄-p₃₄, andN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C with the compounds of Formulae 1 and 2obtained, for example, as described in Examples 1E, 1G, 2C, 10D, 19I,19L, 20B, 20D and 20F, and substituting OVA with the antigenscorresponding to X (or X′ or X″), respectively, it is shown that thecompounds from Examples 1E, 1 G, 2C, 10D, 19I, 19L, 20B, 20D and 20Fmitigate an antigen X-specific immune response after adjuvented antigenX challenge.

Example 17 Pharmacokinetics

17A. OVA and fusion proteins N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C andN-OVA-Gly₃Ser-DOM-Gly₃Ser-6×His-C (prepared, e.g., as described inExample 10) are labeled with IRDye 8000W (LI-COR Biosciences) accordingto manufacturer's instructions. Unreacted dye is removed using Zebadesalting columns (Thermo Scientific). The labeled proteins, 50 μg in100 μl PBS per dose, are administered i.v. or s.c into C57BL/6 mice (5mice per group). At time=0, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 96 hoursafter injection, blood samples are collected from the tip of the tailinto heparin-coated capillary tubes. Samples are store at 4° C.,protected from light, until analysis.

On the day of analysis, blood samples are centrifuged to remove cellularcomponents. Plasma is transferred to fresh capillary tubes and scannedusing an Odyssey Infrared Imaging System (LI-COR Biosciences). Signal isacquired in the 800 nm channel. Using the image-processing programImageJ (US National Institutes of Health), each sample is approximatedas a line of width 2 and the mean intensity along the line is determinedas a relative measure of the amount of circulating protein at that timepoint.

Fluorescence signals (normalized to fluorescence at time=0) vs. time forOVA and fusion proteins DOM-OVA and OVA-DOM, along with curve fits inthe form of bi-exponential decays, in FIG. 8 , show that theASGPR-targeted OVA fusion proteins are cleared from circulation morequickly than unmodified OVA.

17B. By following the procedure described in Example 17A andsubstituting N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C fusion protein and OVAwith asialo-Factor VIII and Factor VIII, respectively, it is shown thatasialo-Factor VIII is cleared from circulation much more quickly thanunmodified Factor VIII.

Example 18 Biodistribution

18A. OVA and N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C fusion protein (prepared,e.g., as described in Example 10) are labeled with Alexa Fluor 647 (LifeTechnologies) according to manufacturer's instructions. Unreacted dye isremoved using Zeba desalting columns (Thermo Scientific). Labeledproteins, 50 μg in 100 μl PBS per dose, are administered i.v. or s.cinto C57BL/6 mice (5 mice per group). Two hours after injection, miceare euthanized by CO₂ asphyxiation. The blood, heart, intestines,kidneys, liver, lungs, stomach, spleen, and remaining carcass are imagedusing an IVIS Spectrum imaging system (Caliper Life Sciences). Data isacquired and analyzed using Living Image software (Caliper LifeSciences). Single cell suspensions of liver and spleen are prepared andstained with fluorescently conjugated antibodies against MHC class II,CD1d, CD3, CD4, CD8a, CD11b, CD11c, CD14, CD19, CD45, CD123, and/or Linin PBS with 0.1% (w/v) BSA. Samples are analyzed using a LSR II flowcytometer and FACS Diva software (BD Biosciences).

A histogram showing fluorescence signal densities (photons/weight) foreach organ and each protein shows that in animals injected withN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C fusion protein the highestfluorescence signal is in the liver as compared to those treated withunmodified OVA.

A histogram showing percentages of N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C andOVA-positive cells shows that animals injected withN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C fusion proteins have a significantlyhigher percentage of positive hepatocytes as compared to those treatedwith unmodified OVA.

18B. By following the procedure described in Example 18A andsubstituting N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C fusion protein and OVAwith asialo-Factor VIII and Factor VIII, respectively, it is shown thatanimals injected with asialo-Factor VIII have a significantly higherpercentage of positive hepatocytes as compared to those treated withunmodified Factor VIII.

18C. By following the procedure described in Example 18A andsubstituting N-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C fusion protein with thecompounds of Formula 1 obtained, for example, as described in Examples3A, 4A, 5B, 6C, 7B and 19G, it is shown that animals injected with thecompounds from Examples 3A, 4A, 5B, 6C, 7B and 19G have a significantlyhigher percentage of positive hepatocytes as compared to those treatedwith OVA.

18D. By following the procedure described in Examples 18A and B, andsubstituting F1aA-OVA-m₄-n₈, F1b-OVA-m₁-n₄-p₃₄, andN-DOM-Gly₃Ser-OVA-Gly₃Ser-6×His-C with the compounds of Formulae 1 and 2obtained, for example, as described in Examples 1E, 1G, 2C, 10D, 19I,19L, 20B, 20D and 20F, and substituting OVA with the antigenscorresponding to X (or X′ or X″), respectively, it is shown that it isshown that animals injected with the compounds from Examples 1E, 1G, 2C,10D, 191, 19L, 20B, 20D and 20F have a significantly higher percentageof positive hepatocytes as compared to those treated with antigen X.

Example 19 F1m-OVA-m₂-n$₀-p₃₀-q₄-CMP-2NHAc

19A. Formula 1102 where R³ is NHAc and R⁴ is OH

N-Acetyl-D-galactosamine (Formula 1101 where R³ is NHAc and R⁴ is OH)(5g, 22.6 mmol) was added to a stirred solution of chloroethanol (200m₁) at room temperature. The solution was cooled to 4° C. andacetylchloride was added drop-wise to the solution. The solution wasbrought to room temperature and then heated to 70° C. After 4 hours, theunreacted choroethanol was removed under reduced pressure. 100 ml ofethanol was added to the crude product and the resulting solution wasstirred in the presence of carbon for 2 hours. The solution wasfiltered, and the solvent was removed under reduced pressure. Thecorresponding product of Formula 1102,N-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide,was used without further purification.

19B. Formula 1103 where R³ is NHAc and R⁴ is OH

TheN-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamideprepared in Example 19A (2g, 7.4 mmol) was added to a stirred solutionof DMF (100 ml) and sodium azide (4g, 61.5 mmol). The solution washeaded at 90° C. for 12 hours and then filtered. The residual solventwas removed under reduced pressure and the crude product was purifiedvia flash chromatography (10% MeOH in dichloromethane) to give thecorresponding product of Formula 1103,N-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide.

19C. Formula 1104 where R³ is NHAc and R⁴ is OH

TheN-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamideprepared in Example 19B (2 g, 6.9 mmol) was added to a solution ofpalladium on carbon and ethanol (50 ml). The solution was stirred underhydrogen gas (3 atm) for 4 hours. The resulting solution was filteredand the residual solvent was removed under reduced pressure to affordthe corresponding product of Formula 1104,N-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide,which was used without further purification.

19D. Formula 1105 where R³ is NHAc and R⁴ is OH

TheN-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamideprepared in Example 19C (1.0 g, 3.78 mmol) was added to a solution ofmethacrylate anhydride (0.583 g, 3.78 mmol) in DMF (50 ml).Triethylamine was then added to the solution and the reaction wasstirred for 2 hours at room temperature. After 2 hours, the excesssolvent was removed under reduced pressure, and the correspondingproduct of Formula 1105,N-(2-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)methacrylamide,was isolated via flash chromatography.

19E. Formula 1107 where p is 30, q is 4, R³ is NHAc, R⁴ is OH and R⁸ isCMP

An azide-modified uRAFT agent of Formula 1106 where q is 4 (28 mg) wasadded to a solution ofN-(2-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)methacrylamideprepared in Example 19D (579 mg, 1.74 mmol) and azobisisobutyronitrile(2.2 mg, 0.0116 mmol) in DMF. The reaction mixture was subjected to 4free-pump-thaw cycles, and then stirred at 70° C. After 12 hours, thepolymer product of Formula 1107, where p is 30 and q is 4 wasprecipitated from the reaction mixture via the addition of methanol. Thesolvent was decanted from the solid and the solid was collected andresidual solvent was removed via reduced pressure.

19F. Formula 1109 where X′ is OVA, m is 2 and n is 80

Ovalbumin (5 mg, 0.00012 mmol) was added to 100 μl of sodium phosphatebuffer (pH 8.0) and stirred. To this solution was added 5 mg of thecompound of Formula 1108 where n is 80. After 1 hour, the unreactedcompound of Formula 1108 was removed from the solution via centrifugalsize-exclusion chromatography. The resulting bufferd solution containingthe corresponding product of Formula 1109 was used in the next reactionwithout further purification.

19G. Formula 1m where X′ is OVA, m is 2, n is 80, p is 30, q is 4, R³ isNHAc and R⁸ is CMP

The solution prepared in Example 19F was added to 100 μl of sodiumphosphate buffer (pH 8.0) which contained 10 mg of the product ofFormula 1107 prepared in Example 19E. The reaction was allowed to stirfor 2 hours and then the excess Formula 1107 was removed via centrifugalsize exclusion chromatography to afford the corresponding isomericproduct of Formula 1 m in solution, which was used in biological studieswithout further purification. The R³ substituent is shown in the name ofthe title compound as 2NHAc.

19H. Other Compounds of Formula 1109

By following the procedure described in Example 19F and substituting OVAwith the following:

-   -   Abciximab,    -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   Insulin (SEQ ID NO:5),    -   GAD-65 (SEQ ID NO:6),    -   IGRP (SEQ ID NO:7)    -   MBP (SEQ ID NO:8),    -   MOG (SEQ ID NO:9),    -   PLP (SEQ ID NO:10),    -   MBP13-32 (SEQ ID NO:11),    -   MBP83-99 (SEQ ID NO:12),    -   MBP111-129 (SEQ ID NO:13),    -   MBP146-170 (SEQ ID NO:14),    -   MOG1-20 (SEQ ID NO:15),    -   MOG35-55 (SEQ ID NO:16),    -   PLP139-154 (SEQ ID NO:17),    -   MART1 (SEQ ID NO:18),    -   Tyrosinase (SEQ ID NO:19),    -   PMEL (SEQ ID NO:20),    -   Aquaporin-4 (SEQ ID NO:21),    -   S-arrestin (SEQ ID NO:22),    -   IRBP (SEQ ID NO:23),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:24),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25),    -   Alpha-gliadin (SEQ ID NO:26),    -   Omega-gliadin (SEQ ID NO:27),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE example (UNIPROT P18577),        there are obtained the following corresponding compounds of        Formula 1109 where n is 80:    -   X is Abciximab and m is 10,    -   X is Adalimumab and m is 11,    -   X is Agalsidase alfa and m is 14,    -   X is Agalsidase beta and m is 14,    -   X is Aldeslukin and m is 6,    -   X is Alglucosidase alfa and m is 13,    -   X is Factor VIII and m is 100,    -   X is Factor IX and m is 18,    -   X is L-asparaginase and m is 5,    -   X is Laronidase and m is 7,    -   X is Octreotide and m is 1,    -   X is Phenylalanine ammonia-lyase and m is 12,    -   X is Rasburicase and m is 12,    -   X is Insulin (SEQ ID NO:5) and m is 2,    -   X is GAD-65 (SEQ ID NO:6) and m is 8,    -   X is IGRP (SEQ ID NO:7) and m is 7,    -   X is MBP (SEQ ID NO:8) and m is 6,    -   X is MOG (SEQ ID NO:9) and m is 5,    -   X is PLP (SEQ ID NO:10) and m is 8,    -   X is MBP13-32 (SEQ ID NO:11) and m is 1,    -   X is MBP83-99 (SEQ ID NO:12) and m is 1,    -   X is MBP111-129 (SEQ ID NO:13) and m is 1,    -   X is MBP146-170 (SEQ ID NO:14) and m is 2,    -   X is MOG1-20 (SEQ ID NO:15) and m is 1,    -   X is MOG35-55 (SEQ ID NO:16) and m is 2,    -   X is PLP139-154 (SEQ ID NO:17) and m is 3,    -   X is MART1 (SEQ ID NO:18) and m is 4,    -   X is Tyrosinase (SEQ ID NO:19) and m is 8,    -   X is PMEL (SEQ ID NO:20) and m is 5,    -   X is Aquaporin-4 (SEQ ID NO:21) and m is 4,    -   X is S-arrestin (SEQ ID NO:22) and m is 12,    -   X is IRBP (SEQ ID NO:23) and m is 21,    -   X is Conarachin and m is 21,    -   X is Alpha-gliadin “33-mer” native (SEQ ID NO:24) and m is 1,    -   X is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25) and m is        1,    -   X is Alpha-gliadin (SEQ ID NO:26) and m is 1,    -   X is Omega-gliadin (SEQ ID NO:27) and m is 1,    -   X is Fel d 1 and m is 4,    -   X is Cat albumin and m is 16,    -   X is Can f 1 and m is 6,    -   X is Dog albumin and m is 23, and    -   X is RhCE example and m is 10.

19I. Other Compounds of Formula 1m

By following the procedure described in Example 19G and substituting thecompounds of Formula 1109, for example as obtained in Example 19H, thereare obtained the following corresponding compounds of Formula 1 m:

-   -   F1m-Abciximab-m₁₀-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Adalimumab-m₁₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Agalsidase alfa-m₁₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Agalsidase beta-m₁₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Aldeslukin-m₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alglucosidase alfa-m₁₃-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Factor VIII-m₁₀₀-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Factor IX-m₁₈-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-L-asparaginase-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Laronidase-m₇-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Octreotide-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Phenylalanine ammonia-lyase-m₁₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Rasburicase-m₁₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Insulin-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-GAD-65-m₈-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-IGRP-m₇-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP-m₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MOG-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-PLP-m₈-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP13-32-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP83-99-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP111-129-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MBP146-170-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MOG1-20-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MOG35-55-m₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-PLP139-154-m₃-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-MART1-m₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Tyrosinase-m₈-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-PMEL-m₅-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Aquaporin-4-m₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-S-arrestin-m₁₂-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-IRBP-m₂₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Conarachin-m₂₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alpha-gliadin “33-mer” native-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alpha-gliadin “33-mer” deamidated-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Alpha-gliadin-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Omega-gliadin-m₁-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Fel d 1-m₄-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Cat albumin-m₁₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Can f 1-m₆-n₈₀-p₃₀-q₄-CMP-2NHAc,    -   F1m-Dog albumin-m₂₃-n₈₀-p₃₀-q₄-CMP-2NHAc, and    -   F1m-RhCE-m₁₀-n₈₀-p₃₀-q₄-CMP-2NHAc.

19J. Formula 1107 where p is 30, q is 8, R³ is OH, R⁴ is OH and R⁸ isCMP

By following the procedure described in Example 19A and substituting theN-acetyl-D-galactosamine with galactose, and following through to theprocedure desribed in Example 19E except using an azide-modified uRAFTagent of Formula 1106 where q is 8, there is obtained the compound ofFormula 1107 where p is 30, q is 8, R³ is OH, R⁴ is OH and R⁸ is CMP.

19K. Formula 1109 where n is 62 and where X′ and m are as in Example 19H

By following the procedure described in Example 19F, substituting theOVA with the compounds as described in Example 19H and employing thecompound of Formula 1108 where n₁ is 62, there are obtained thecorresponding compounds of Formula 1109 where n is 62.

19L. Other Compounds of Formula 1 m

By following the procedure described in Example 19G and substituting thecompound of Formula 1107 with the compounds obtained in Example 19J, andsubstituting the compound of Formula 1109 with the compounds obtained inExample 19K, there are obtained the following corresponding compounds ofFormula 1 m:

-   -   F1m-Abciximab-m₁₀-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Adalimumab-m₁₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Agalsidase alfa-m₁₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Agalsidase beta-m₁₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Aldeslukin-m₆-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alglucosidase alfa-m₁₃-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Factor VIII-m₁₀₀-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Factor IX-m₁₈-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-L-asparaginase-m₅-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Laronidase-m₇-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Octreotide-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Phenylalanine ammonia-lyase-m₁₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Rasburicase-m₁₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Insulin-m₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-GAD-65-m₈-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-IGRP-m₇-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-MBP-m₆-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-MOG-m₅-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-PLP-m₈-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP13-32-m₁-n₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP83-99-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP111-129-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-MBP146-170-m₂-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-MOG1-20-m₁-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-MOG35-55-m₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-PLP139-154-m₃-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-MART1-m₄-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-Tyrosinase-m₈-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-PMEL-m₅-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-Aquaporin-4-m₄-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-S-arrestin-m₁₂-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-IRBP-m₂₁-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-Conarachin-m₂₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alpha-gliadin “33-mer” native-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alpha-gliadin “33-mer” deamidated-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Alpha-gliadin-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Omega-gliadin-m₁-n₆₂-p₃₀-q₈-CMP-2OH,    -   F1m-Fel d 1-m₄-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-Cat albumin-m₁₆-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-Can f 1-m₆-n₆₂-p₃₀-q-CMP-2OH,    -   F1m-Dog albumin-m₂₃-n₆₂-p₃₀-q-CMP-2OH, and    -   F1m-RhCE-m₁₀-n₆₂-p₃₀-q₈-CMP-2OH.

Example 20 F1n-insulin-m₂-n₁-p₃₀-q₄-CMP-2NHAc

20A. Formula 1202 where X′ is Insulin, m is 2 and n is 1

Recombinant human insulin (5 mg) was added to 100 μl of DMF containing10 μl of triethylamine and stirred until the insulin became soluble. Tothis solution was added 10 mg (0.0161 mmol) of a linker precursor ofFormula 1201 where n is 1 and the reaction was allowed to stir. After 1hour, 1.3 ml of tert-butyl methyl ether was added to isolate thecorresponding product of Formula 1202, which was recovered as theprecipitate. Residual DMF and tert-butyl methyl ether were removed underreduced pressure. Characterization via liquid chromatography, massspectroscopy and polyacrylamide gell electrophoresis confirmed theidentity of the product. The modified insulin product of Formula 1202was used without further purification.

20B. Formula 1 n where X′ is Insulin, m is 2, n is 1, p is 30, q is 4and R⁸ is CMP

The product of Formula 1202 obtained in Example 20A was resuspended in100 μl of DMF. The poiymer product of Formula 1107 obtained in Example19E (10 mg) was added and the reaction was allowed to stir for 1 hour.After 1 hour, the reaction products were precipitated via the additionof dichloromethane (1.3 ml). The product was filtered and the residualsolvent was removed under reduced pressure. The crude product was thenresuspended in 500 μl of PBS, and the low molecular weight componentswere removed via centrifugal size exclusion chromatography to afford thecorresponding isomeric product of Formula 1 n. Characterization vialiquid chromatography, mass spectroscopy and polyacrylamide gellelectrophoresis confirmed the identity of the product. The modifiedinsulin product of Formula 1202 was used without further purification.

20C. Other Compounds of Formula 1202

By following the procedure described in Example 19F and substituting OVAinsulin the following:

-   -   Abciximab,    -   Adalimumab,    -   Agalsidase alfa,    -   Agalsidase beta,    -   Aldeslukin,    -   Alglucosidase alfa,    -   Factor VIII,    -   Factor IX,    -   L-asparaginase,    -   Laronidase,    -   Octreotide,    -   Phenylalanine ammonia-lyase,    -   Rasburicase,    -   GAD-65 (SEQ ID NO:6),    -   IGRP (SEQ ID NO:7)    -   MBP (SEQ ID NO:8),    -   MOG (SEQ ID NO:9),    -   PLP (SEQ ID NO:10),    -   MBP13-32 (SEQ ID NO:11),    -   MBP83-99 (SEQ ID NO:12),    -   MBP111-129 (SEQ ID NO:13),    -   MBP146-170 (SEQ ID NO:14),    -   MOG1-20 (SEQ ID NO:15),    -   MOG35-55 (SEQ ID NO:16),    -   PLP139-154 (SEQ ID NO:17),    -   MART1 (SEQ ID NO:18),    -   Tyrosinase (SEQ ID NO:19),    -   PMEL (SEQ ID NO:20),    -   Aquaporin-4 (SEQ ID NO:21),    -   S-arrestin (SEQ ID NO:22),    -   IRBP (SEQ ID NO:23),    -   Conarachin (UNIPROT Q6PSU6),    -   Alpha-gliadin “33-mer” native (SEQ ID NO:24),    -   Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25),    -   Alpha-gliadin (SEQ ID NO:26),    -   Omega-gliadin (SEQ ID NO:27),    -   Fel d 1A (UNIPROT P30438),    -   Cat albumin (UNIPROT P49064),    -   Can f 1 (UNIPROT 018873),    -   Dog albumin (UNIPROT P49822), and    -   RhCE example (UNIPROT P18577),        there are obtained the following corresponding compounds of        Formula 1202 where n is 1:    -   X is Abciximab and m is 10,    -   X is Adalimumab and m is 11,    -   X is Agalsidase alfa and m is 14,    -   X is Agalsidase beta and m is 14,    -   X is Aldeslukin and m is 6,    -   X is Alglucosidase alfa and m is 13,    -   X is Factor VIII and m is 100,    -   X is Factor IX and m is 18,    -   X is L-asparaginase and m is 5,    -   X is Laronidase and m is 7,    -   X is Octreotide and m is 1,    -   X is Phenylalanine ammonia-lyase and m is 12,    -   X is Rasburicase and m is 12,    -   X is GAD-65 (SEQ ID NO:6) and m is 8,    -   X is IGRP (SEQ ID NO:7) and m is 7,    -   X is MBP (SEQ ID NO:8) and m is 6,    -   X is MOG (SEQ ID NO:9) and m is 5,    -   X is PLP (SEQ ID NO:10) and m is 8,    -   X is MBP13-32 (SEQ ID NO:11) and m is 1,    -   X is MBP83-99 (SEQ ID NO:12) and m is 1,    -   X is MBP111-129 (SEQ ID NO:13) and m is 1,    -   X is MBP146-170 (SEQ ID NO:14) and m is 2,    -   X is MOG1-20 (SEQ ID NO:15) and m is 1,    -   X is MOG35-55 (SEQ ID NO:16) and m is 2,    -   X is PLP139-154 (SEQ ID NO:17) and m is 3,    -   X is MART1 (SEQ ID NO:18) and m is 4,    -   X is Tyrosinase (SEQ ID NO:19) and m is 8,    -   X is PMEL (SEQ ID NO:20) and m is 5,    -   X is Aquaporin-4 (SEQ ID NO:21) and m is 4,    -   X is S-arrestin (SEQ ID NO:22) and m is 12,    -   X is IRBP (SEQ ID NO:23) and m is 21,    -   X is Conarachin and m is 21,    -   X is Alpha-gliadin “33-mer” native (SEQ ID NO:24) and m is 1,    -   X is Alpha-gliadin “33-mer” deamidated (SEQ ID NO:25) and m is        1,    -   X is Alpha-gliadin (SEQ ID NO:26) and m is 1,    -   X is Omega-gliadin (SEQ ID NO:27) and m is 1,    -   X is Fel d 1 and m is 4,    -   X is Cat albumin and m is 16,    -   X is Can f 1 and m is 6,    -   X is Dog albumin and m is 23, and    -   X is RhCE example and m is 10.

20D. Other Compounds of Formula 1 n

By following the procedure described in Example B and substituting thecompounds of Formula 1202, for example as obtained in Example 20C, thereare obtained the following corresponding compounds of Formula 1 m:

-   -   F1n-Abciximab-m₁₀-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Adalimumab-m₁₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Agalsidase alfa-m₁₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Agalsidase beta-m₁₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Aldeslukin-m₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alglucosidase alfa-m₁₃-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Factor VIII-m₁₀₀-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Factor IX-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-L-asparaginase-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Laronidase-m₇-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Octreotide-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Phenylalanine ammonia-lyase-m₁₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Rasburicase-m₁₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-GAD-65-m₈-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-IGRP-m₇-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP-m₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MOG-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-PLP-m₈-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP13-32-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP83-99-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP111-129-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MBP146-170-m₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MOG1-20-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MOG35-55-m₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-PLP139-154-m₃-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-MART1-m₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Tyrosinase-m₈-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-PMEL-m₅-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Aquaporin-4-m₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-S-arrestin-m₁₂-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-IRBP-m₂₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Conarachin-m₂₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alpha-gliadin “33-mer” native-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alpha-gliadin “33-mer” deamidated-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Alpha-gliadin-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Omega-gliadin-m₁-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Fel d 1-m₄-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Cat albumin-m₁₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Can f 1-m₆-n₁-p₃₀-q₄-CMP-2NHAc,    -   F1n-Dog albumin-m₂₃-n₁-p₃₀-q₄-CMP-2NHAc, and    -   F1n-RhCE-m₁₀-n₁-p₃₀-q₄-CMP-2NHAc.

20E. Formula 1202 where n is 33 and where X′ and m are as in Example 20C

By following the procedure described in Example 19F, substituting theinsulin with the compounds as described in Example 20C and employing thecompound of Formula 1201 where n is 33, there are obtained thecorresponding compounds of Formula 1202 where n is 33.

20F. Other Compounds of Formula 1 n

By following the procedure described in Example 20B and substituting thecompound of Formula 1107 with the compounds obtained in Example 19J, andsubstituting the compound of Formula 1202 with the compounds obtained inExample 20E, there are obtained the following corresponding compounds ofFormula 1n:

-   -   F1n-Abciximab-m₁₀-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Adalimumab-m₁₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Agalsidase alfa-m₁₄-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Agalsidase beta-m₁₄-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Aldeslukin-m₆-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alglucosidase alfa-m₁₃-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Factor VIII-m₁₀₀-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Factor IX-m₁₈-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-L-asparaginase-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Laronidase-m₇-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Octreotide-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Phenylalanine ammonia-lyase-m₁₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Rasburicase-m₁₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-GAD-65-m₈-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-IGRP-m₇-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-MBP-m₆-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-MOG-m₅-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-PLP-m₈-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-MBP13-32-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP83-99-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP111-129-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MBP146-170-m₂-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-MOG1-20-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-MOG35-55-m₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-PLP139-154-m₃-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-MART1-m₄-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Tyrosinase-m₈-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-PMEL-m₅-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Aquaporin-4-m₄-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-S-arrestin-m₁₂-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-IRBP-m₂₁-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Conarachin-m₂₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alpha-gliadin “33-mer” native-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alpha-gliadin “33-mer” deamidated-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Alpha-gliadin-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Omega-gliadin-m₁-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Fel d 1-m₄-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Cat albumin-m₁₆-n₃₃-p₃₀-q₈-CMP-2OH,    -   F1n-Can f 1-m₆-n₃₃-p₃₀-q-CMP-2OH,    -   F1n-Dog albumin-m₂₃-n₃₃-p₃₀-q₈-CMP-2OH, and    -   F1n-RhCE-m₁₀-n₃₃-p₃₀-q₈-CMP-2OH.

Example 21 Clearance of OVA-Specific Antibodies from Circulation

To induce production of OVA-specific antibodies for subsequentdepletion, C57BL/6 mice were injected i.v. with up to five doses of 10μg OVA until a titer of anti-OVA IgG in plasma, as determined by ELISA,reached 3 (FIG. 9A). Plasma samples were prepared by centrifugation ofblood collected into EDTA-coated tubes at 2000×g for 10 minutes at roomtemperature and stored at −20° C. until analysis. Titer is defined asthe log₁₀ of the maximal fold dilution of plasma with detectableanti-OVA IgG. Here, plasma samples were assayed at dilutions of 10-,50-, 100-, 500-, 1,000-, 5,000-, 10,000-, and 50,000-fold, yieldingpotential titer measurements of 0, 1, 1.7, 2, 2.7, 3, 3.7, 4, and 4.7.Mice with titers below 3 after five doses of 10 μg OVA were given twoadditional doses of 10 μg OVA adjuvanted with 10 μg CpG-B to boostanti-OVA IgG production and hence the amount of circulating anti-OVA IgGavailable for depletion (FIG. 9B).

To evaluate the efficacy of hepatocyte ASGPR-targeted OVA for anti-OVAantibody clearance, mice with circulating anti-OVA IgG were injectedi.v. with saline or saline containing molar equivalents of DOM and OVA,DOM-OVA, or OVA-DOM. In particular, mice were treated with molarequivalents of 10, 50, 100, or 200 μg OVA, as indicated in FIG. 9A-9B.At times t=−1 day, +3 hours, +1 day, and +7 days, relative to the timeof treatment injection, blood samples were collected to assess, by ELISAanalysis of plasma, the amount of anti-OVA IgG in circulation. Clearanceof anti-OVA IgG from circulation is expected to decrease the amount ofanti-OVA IgG in plasma and hence decrease plasma anti-OVA IgG titer asdetermined by ELISA. In mice immunized with OVA alone and treated withDOM-OVA or OVA-DOM, titers of anti-OVA IgG decreased by up to 3 or 2,respectively, representing decreases in the amount of circulatinganti-OVA IgG by 1000- or 100-fold, which is up to 50-fold more effectivethan control treatment using saline or DOM and OVA (FIG. 9A). In miceimmunized with OVA adjuvanted with CpG-B, clearance of circulatinganti-OVA IgG was not observed (FIG. 9B).

Example 22 NOD Mouse

Non-obese diabetic (NOD) mice are susceptible to the spontaneous onsetof autoimmune diabetes mellitus, which is the result of an autoimmuneresponse to various pancreatic auto-antigens. Diabetes develops in NODmice as a result of insulitis, characterized by the infiltration ofvarious leukocytes into the pancreatic islets.

In order to evaluate the efficacy of a treatment for diabetes mellitus,starting at 6-weeks of age, NOD mice are divided into control or testgrops and treated, respectively, with weekly intravenous injections of atest composition (10 μg) or an inactive control such as saline. Theinjections continue for 18 consecutive weeks.

The blood glucose concentration of the mice is measured weekly. Micethat maintain a blood glucose concentration of less than 300 mg/mlduring the experiment are considered non-diabetic. In addition, at theend of the study the pancreases of the mice are harvested and T cellinfiltration in the pancreas is determined via immunohistochemistry asan assessment of insulitis. Tolerance induction is assessed by thedepletion of auto-antigen specific CD8 T cells as compared to mice thatare treated with saline and develop diabetes. The existence ofauto-antigen specific CD8 T cells is determined via ELISpot assay.

When tested as described above, compositions of Formulae 1 and 2 where Xis insulin, such as F1m-insulin-m₂-n₁-p₃₀-q₄-2NAcGAL, show efficacy fortreating diabetes mellitus.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes can be made and equivalents can besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications can be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the claims appended hereto. All patents and publications cited aboveare hereby incorporated by reference.

1. A compound for the induction of antigen-specific immune tolerance ina subject, the compound comprising: a peptide to which tolerance isdesired; wherein the peptide to which tolerance is desired, whenpresented to the subject alone, is capable of inducing an unwantedimmune response in the subject; a polymeric linker; wherein thepolymeric linker is coupled to the peptide to which tolerance is desiredvia a disulfide bond or a disulfanyl ethyl ester; wherein the disulfidebond or the disulfanyl ethyl ester are each configured to be cleavedupon administration of the compound to the subject and to release thepeptide to which tolerance is desired from the polymeric linker; whereinthe polymeric linker comprises a 1-cyano-1-methyl-propyl group andmethacrylic units comprising an ethylacetamido functionality; and aliver-targeting moiety; wherein the liver-targeting moiety comprises agalactosylating moiety; wherein the liver-targeting moiety is coupled tothe polymeric linker through the ethylacetamido functionality.